Respiratory infections may reflect deficiencies in host defense mechanisms

is Professor of Medicine and Head of the Pulmonary Section, Department of Medicine, Yale University School of Medicine, New Haven. Dr. Reynolds is a graduate of the University of Virginia School of Medicine and completed his medical training at the New York Hospital-Cornell Medical Center and the University of Washington, Seattle. His research training in pulmonary immunology and lung infections was done in the Laboratory of Clinical Investigation of the National Institute of Allergy and Infectious Diseases, NIH, Bethesda, and these research interests have been continued at Yale. His special interest focuses on the use of bronchoalveolar lavage for analysis of cells and proteins (immunoglobulins) in normal humans to study lung host defense mechanisms and alveolar macrophage function. The control of lung inflammation has been a special research area. Dr. Reynolds is a subspecialist of the American Board of Allergy and Immunology and a member of the American Society of Clinical Investigation.

CHRONIC RESPIRATORY DISEASE includes an enormous number of derangements that can affect any portion of the respiratory tract-upper airways (nose, sinuses, and oropharnyx), conducting airways (trachea down to the respiratory bronchioles), the air exchange surface of the al-

6

veoli, interstitium, or architectural support of the air sacs and pulmonary vasculature. Of course, not every form of chronic respiratory disease is associated with increased susceptibility to infection, but infection is often an intimate part of the process or a complicating factor that must be controlled. Exposure to a virulent microorganism or to a large inoculum, if inhaled or aspirated into the lungs, may cause illness in even a normal person; but in many of the cases in which infection is a problem, the general apparatus at fault can be the pulmonary host defense system. Some malfunctioning or compromised component of this intricate defense system often can be correlated with a propensity for infection (Table 1). For example, an endotracheal tube allows direct access to the lung, bypassing the larynx and the other upper airway structures. If ciliary clearance is impaired by structural defects in cilia located on the apical edge of the airway epithelial lining cells, removal of mucus and respiratory secretions is sluggish and ineffective. If the postoperative patient, perhaps because of abdominal pain or depressed consciousness, cannot cough well, secretions accumulate in the airways. In patients with hypogammaglobulinemia or dysgammaglobulinemia, the absence of opsonic antibody can foster infections with encapsulated bacteria. Cytotoxic antineoplastic chemotherapy and other forms of immunosuppression can produce granulocytopenia, which prevents mobilization of an inflammatory reaction. All of these deficiencies in pulmonary host defense, whether acquired or inherent, are associated with obvious syndromes of respiratory infection. Moreover, the normal person without antecedent lung disease is equally at risk for the development of infection under some of the circumstances just discussed. In adults, the reason for having a respiratory tract infection is often obvious: chronic bronchitis, aggravated by cigarette smoking, or chronic obstructive lung disease. Occasionally, however, the physician is confronted with a relatively young person who has an unexpected number of respiratory problems which seem inappropriate. The problem may not be an overt lung infection. It may be a cough and excessive sputum, poorly controlled allergic rhinitis, asthma, frequent sinusi-

TABLE

l.-LUNG

HOST

HOST MECHANISMS

Mechanical etc.)

barriers

Mucociliary

clearance

POTENTIAL

(larynx,

(cough)

Bronchoconstriction

Iron-containing (transferrin,

IgA

proteins lactoferrin)

Alternative pathway

complement activation

classes

AIRWAY

DEFECT

CHALLENGES IMPACT

AND

POTENTIAL

INFECTION

Aspiration, direct microorganisms

Intrinsic Ciliotoxic

Stagnant secretions, coughing, bronchiectasis, sinusitis

structural infections airways,

defect

m cilia

intrinsic

aerosol entry into airway

Sinopulmonary infections Abnormal colonization with bacteria

Iron

May not inhibit (Pseudomonas,

certain bacteria E. coli)

Sinopulmonary Pneumonia bacteria

infections encapsulated

deficiency

Acquired Selective C3 and

certain

Milieu

hypogammaglobulinemia IgG4 and IgGc deficiency C5 deficiency

of

Poor removal of secretions Excessive secretions

IgA deficiency Functional deficiency from breakdown by bacterial IgAi proteases

Alveolar Other immunoglobulin (opsonic IgG)

FOR

Conducting Airways Bypassing barriers with an endotracheal tube or tracheostomy

Hyperactive asthma

Local immunoglobulin coating-secretory

DEFENSES

Trouble with threatening

with

infection

but

not

life-

Surfactant Alveolar

macrophages

Decreased synthesis Acute lung injury

Loss of opsonization Alveolar collapse

Subtle effects from immunosuppression Cannot kill intracellular

Propensity for Pneumocystis carinii and Legionella infections; poor containment of mycobacterium

microbes

activity (atelectasis)

Polymorphonuclear granulocytes

Absent because of immunosuppression: Intrinsic defects of motility Lack of chemotacic stimulus

Initiation of immune responses (humoral antibody and cellular immunity1

Immunosuppression

Inadequate S-IgA or IgG antibody available (more susceptible to viral, mycoplasmal, and bacterial infections)

Generation of an inflammatory response (influx of PMN granulocytes, eosinophils, lymphocytes, and fluid components)

Generally supply Impaired

Same as for PMN granulocytes. C5 deficiency might decrease inflammatory response

Augmenting

Poor inflammatory response, associated with gram-negative bacillary infection and fungi (Aspergillus)

Mechanisms

reflects status and of PMN granulocytes adherence

tis, recurrent nasal polyps, and bouts of otitis media. Because the severity of these respiratory problems may not seem great, they may not readily alert the physician that something unusual is present. But the physician should be prepared to evaluate this type of patient thoroughly. A propensity for infection may not have been obvious in childhood but became apparent in the patient’s teens or early 20s. Because genetic defects usually manifest in infancy, this patient’s situation may falsely suggest that the defect is not genetic. That assessment may not be accurate, because subtle or minor forms of host deficiency disease are being increasingly recognized in adults. Recurrent respiratory infections often provide the clue to them. This monograph presents information on subtle abnormalities in lung host defenses that either can explain infection in the patient with existing chronic lung disease or can cause lung disease directly. By examining some of the potential host defects that predispose to microbial colonization and infection, we hope new ideas about protecting the susceptible person will emerge and will lead to new therapies and strategies for fortifying host defense mechanisms. Pneumonia, the most serious consequence of respiratory infection, ranks as the fifth cause of death in the United States and remains a major medical problem. A combination of upper respiratory tract infections and lower airway infections (bronchitis or pneumonia) indicates the possibility of several immunodeficiency syndromes that are not found exclusively in the pediatric age group but can exist in young adults as mild, heterozygous, nonlethal forms of disease. The possible diseases include cystic fibrosis, defects in the mucociliary clearance apparatus or immotile ciliary syndromes, acquired hypogammaglobulinemia, and dysfunction of phagocytic cell killing or motility (responsiveness to chemoattractant stimuli). A detailed patient history can immediately provide important information about affected siblings, infertility, or a striking change in respiratory health that makes an acquired abnormality likely. Preliminary screening tests also are indicated and will be discussed. 10

LUNG

HOST DEFENSES

The atmosphere we breathe is not simply “air” but represents a complex mixture of gases and particulates. To this ambient air are further added virus- and bacteria-containing droplets, found in respiratory secretions coughed and sneezed by our immediate neighbors, which can be aerosolized into our airways. Moreover, we frequently aspirate secretions from our upper respiratory tract, particularly during sleep. To perform air exchange adequately, the respiratory system must recognize and eliminate these unwanted elements in inspired air. This nonrespiratory activity of purifying inspired air and keeping lung tissues free of infection has been collectively termed lung host defense. Considerable interest in this aspect of respiratory physiology has developed, and several reviews provide many details. 1-7 Components of the defense system are spaced along the entire respiratory tract from the point of air intake at the nares to the level of oxgen uptake on the alveolar surface. Conducting airways functionally extend from the nose to the respiratory bronchioles. In this segment of the airways, nasal turbinates, the epiglottis, larynx, and other anatomical barriers exist. Dichotomous branching of the respiratory tree causes the air stream to deflect and particles carried in it to impact on the mucosal surface. Entrapment in mucus and other locally derived proteins (such as secretory IgA), ciliary clearance, and coughing further serve to remove particulate material from the respiratory tract. Distal to the respiratory bronchioles in the air exchange units, other components of host defense become important. The lining material of the alveoli (surfactant), iron-containing proteins (transferrin), other immunoglobulins such as IgG opsonins, or properdin (factor B), which may initiate the alternate pathway of complement activation-all have varying activity against inhaled particles or microorganisms. Finally, alveolar macrophages are the principal phagocytic cells in the air spaces and scavengers of the alveolar surfaces. All of the host defense components listed are operant in the normal human respiratory tract and may be categorized as ongoing surveillance mechanisms. 11

Either their function is mechanical, or they may be activated by nonspecific or nonimmunologic stimuli. In addition, several augmenting mechanisms exist that enhance the responsiveness of the system and make it flexible and adaptable. One of these is the lung’s ability to mount immune responses (humoral and cellular) to various antigenic stimuli that bombard the organ. Another is the potential to mount readily an inflammatory response in the lung parenchyma, which allows components in plasma and blood cells to bolster the local resources of the lung. In summary, coordinated function of lung defense mechanisms along the respiratory tract is remarkably effective in removing or neutralizing microorganisms, particulates, and noxious gases that are inhaled with respired air or aspirated in nasopharyngeal secretions. Lung infections and overt harm from air pollutants are rare for the healthy human, considering our constant exposure to airborne substances in ambient air and those emanating from fellow humans who cough and sneeze, or to one’s own indigenous bacterial flora. This defense system can be divided into the components of surveillance mechanisms, which work constantly and in a reflex manner to cleanse the airways, and augmenting mechanisms, which give backup support in unusual situations but require more time or complexity to activate. Many of the mechanical barriers and reflex actions are concentrated in the nose, oropharynx, and along the conducting airways, and the combination of adherence to the mucosal surface, mucociliary clearance, and coughing removes the bulk of debris. What evades these mechanisms and alights on the alveolar surface is efficiently consumed by roaming macrophages, which are assisted by other components of the humoral immune system and various lipoproteins and glycoproteins mixed in the alveolar lining fluid (Fig 1). In the event that local phagocytes and alveolar defenses are inadequate, the inflammatory reaction can be initiated, which unleashes the potent polymorphonuclear neutrophils (PMNs), complement factors, and other vasomediators and humoral immune elements from systemic sources. Prior contact with a microbial agent or some sensitizing substance in the airways may induce immunity 12

Mechanical FaCtOrS ,

Muco-cillary Transport

\ AERODYNAMIC FILTRATION

Fig l.-Factors responsible for clearance of bacteria (6) inhaled into the lungs are quite different in the upper respiratory tract (URT) and in the lower respiratory tract, here represented by enlargement of an alveolus. A bacterium of critical size that escapes mechanical removal from the URT and is deposited in an alveolus may encounter surfactant (secreted by Type II epithelial cells) and/or immunoglobulins (antibodies) (secreted by B-lymphocytes or plasma cells) and complement proteins which condition it for phagocytosis by a resident alveolar macrophage (AM). Antibody with specific opsonizing potential can facilitate attachment of the bacterium to the AM surface membrane through specialized cell receptors. A complement component (C3) may augment such attachment. At least two alternative mechanisms can be activated to enhance killing and clearance of the microbe. First, the AM can liberate chemotactic factors that attract nearby polymorphonuclear phagocytes (PMNs), marginated in a lung capillary adjacent to the alveolus, and thus initiate an inflammatory response. Second, the bacterium may trigger immune lymphocytes (T Iym) to release effector substances (lymphokines) which may activate or stimulate AM phagocytic and bactericidal capacity.

13

which specific antibody (secretory IgA to prevent mucosal adherence or an opsonin to facilitate phagocytosis) may help the lung deal more efficiently with it on rechallenge at a future time. through

NOSE

AND

OROPHARYNX

These areas are the normal channels for inhaled air to reach the glottis and pass into the extrathoracic portion of the trachea before it enters the thorax. With nasal breathing, air is partially conditioned for correct humidity and temperature as it flows over the nasal turbinates and mucosa into the posterior pharynx. Because of nasal obstruction or ventilation requirements for exercise and exertion (usually breathing at more than 20-30 L per minute)? mouth breathing becomes essential, but inhaled air may have to pass well into the trachea before appropriate climatic conditions are obtained. This distance that air must travel before it is warmed appropriately for the alveolar surface has been studied in humans with airway-placed thermistors’ and is very dependent on the original ambient air conditions. In the upper airway, several transitions in the epithelial cell covering occur. In the nares, stratified squamous epithelium is present which gradually changes to a pseudostratified, columnar, ciliated, mucus-secreting epithelium over the nasal turbinates that extends into the posterior pharynx. In the oropharynx, squamous epithelium is present and lines the cavity down to the epiglottis and larynx; below the vocal cords the typical respiratory ciliated epithelium reappears to surface the trachea, major bronchi, and other conducting airways that lead to the respiratory bronchioles. The nasal mucosa is very vascular and has great potential to add copious amounts of fluid to the surface; mucus secretion is plentiful, also. In the mouth, as fluid is basically required to masticate food and to be a lubricant of sorts for the tongue and larynx, its origin is in the parotid and salivary glands, where amylase and sialic acid are provided. This fluid is of minor importance in the air-conditioning process. Host defense mechanisms in the nose and mouth that filter contaminants in the air and control indigenous bacteria, which are plentiful 14

in both areas, are very different but have some similar features, too. In the nose, sneezing (or blowing) is the counterpart of coughing and provides a high-velocity ejection from the nares of any pollen or large-sized particle (>lO IL in diameter) that is an irritant or a nuisance there. Coarse hairs in the nares also facilitate exclusion of gross particles or their entrapment in mucus. For substances that attach to the mucosa, the response of the nose to exude large quantities of watery secretions to wash off its surface is possibly the most effective means, although a person with hay fever or vasomotor rhinitis may not agree. Mucociliary clearance is operant also. Downspouts leading from the ears and lacrimal glands and from sinus cavities provide multiple points for more fluid to be added to the nasal secretions but also create vulnerable points that can make the gutters stop up. The complex plumbing in the nose works if there is good gravitational flow and the orifices stay open; if not, sinusitis, otitis media, and occluded tear ducts result. Substances in nasal secretions that control bacteria or viruses have received most attention, and perhaps Dr. Alexander Fleming’s characterization of the bacteriolytic enzyme lysozymeg initiated research on defense mechanisms in the upper respiratory tract. With the contemporary research effort into the secretory immune system, which began in the early 1960s and focused on the role of immunoglobulins, especially that of secretory IgA (S-IgA),1°-13 the nose was the first portion of the respiratory tract to be investigated (parotid gland secretions are considered part of the digestive tract). Several articles14P17 reported that nasal secretions, like those from other external or mucosal surfaces, were rich in IgA which was synthesized locally by submucosal plasma cells. IgA accounts for about 10% of the total protein content in nasal wash. IgG and siderophilin are present in smaller amountsi and IgE probably is not secreted by nonatopic normal people, although its detection in nasal wash has been controversial.” As an example, in lo- to 20-ml specimens of nasal washings from normals, which were concentrated fivefold, we measurd about 0.5 mg of total protein; this protein consisted of 15% albumin, 15% S-IgA, about 1% IgG, and almost no 1gE.i’ Free secretory 15

component (SC) can be detected in nasal wash fluid.” Of the nasal immunoglobulins, S-IgA is the major source of antibody; only in people with allergic rhinitis will IgE antibody be substantial. The specificity of IgA antibody usually is antiviral. Many articles (reviewed in detail by Tomasi13 and Reynolds and Merril12i) note that after nasal immunization of normal subjects with various viral or mycoplasmal vaccines, appropriate neutralizing IgA antibody can be elicited. Although these antibodies are protective against homologous and live microbial challenge, the duration of protection is often brief and the antibody titers diminish rapidly unless repeated exposure occurs. Certainly this antibody system is a major host defense mechanism in the upper airway, but it is still poorly understood and has proved inordinately difficult for investigators to harness and to manipulate in a predictable manner. The dream that a number of good nasal viral vaccines could be used broadly in the population to immunize against common agents, thus reducing one of the greatest causes of minor morbidity and time lost from work, seems to be fading away. Success with certain bacterial vaccines may be more promising. In the mouth, the sweeping action of the tongue against all surfaces during chewing, swallowing, and expectorating should make it difficult for bacteria to stay in place and to stick. Yet bacteria adhere to buccal squamous cells, and many accumulate in crevices around teeth and gums. Many kinds are present-aerobes and anaerobes, spirochetes, gram-positive and gram-negative species, and some that specialize in making dental plaque and decaying teeth. A common feature of host defense in the mouth and nose is the plentiful amount of S-IgA in secretions that bathe each area. The parotid glands, and probably the submandibular salivary glands secrete IgA as their principal humoral immune substance, which accounts for 12%-15% of the total protein content. In this fluid, albumin represents about 10% of the protein, but IgG is barely detectable (>l%). In parotid fluid IgA is found in monomeric and dimerous forms, and free secretory component can be detected as we1L2’ Thus, normal nasal and parotid (or sali16

vary) secretions have about the same composition of immunoglobulins. Crevicular fluid that seeps from the edge of the gum resembles an ultrafiltrate of plasma and has a high total protein which is mainly IgG, but contains a little IgA. As with the nasal immune system, it has been possible to manipulate S-IgA in the mouth and to produce antibodies against certain cariogenic strains of streptococci that will interfere with bacterial adherence to teeth.223 23 We cannot review this subject in detail here, but substantial success can be achieved in preventing caries with intrasalivary gland immunization in animal models, As for human host defenses, regular brushing and flossing, coupled with some fluoride in the water supply and moderation of candy or sugar in the diet, may still be the best solution. Two reasons for including a section on the nose and mouth in a review of respiratory infections are (1) to remind us that there is an “upper portion” of the respiratory tract that has some features in common with the lower part, particularly the mucosal surface, and (2) to emphasize that infections in the nose, sinuses, ears, and teeth and gums are common and often have ramifications for the diagnosis or successful treatment of illness in the lower respiratory tract. Aspiration of anaerobic bacteria in oral secretions contributes to lung abscess formation, whereas chronic sinusitis often accompanies such diseases as cystic fibrosis, ciliary dyskinetic syndromes, asthma, and dysgammaglobulinemia. Acute viral infections of the nose may be a prelude to bacterial lung infection, and various allergic diseases can simultaneously cause rhinitis and symptoms of hyperactive airways (asthma syndrome). Control of asthma symptoms may be very difficult unless an unrecognized sinus infection is discovered and treated. The presence of nasal polyps in an asthmatic may be expected, but their development in a child with recurrent respiratory infections must raise the possibility of cystic fibrosis. Chronic aspiration pneumonias in a neurologic patient with an incompetent larynx may necessitate separating completely the upper and lower tracts by oversewing the vocal cords and by the placement of a tracheostomy. 17

CONDUCTING

AIRWAYS

Within this segment of the respiratory tract, situated between the upper airway (nose, oropharynx, and larynx) and the air exchange area of the terminal bronchioles and alveoli, mucociliary clearance and coughing are the principal means of cleansing the mucosal surface. S-IgA and other antibodies probably prevent epithelial attachment of certain bacteria and viruses as well to the ciliated cells. Multiple branching points of the respiratory tree increases impaction of airborne particulates against the mucosa at high velocity. Because many respired particles (< 5 > 1 lo in size) are filtered out in the conducting airways, host defenses must be efficient. However, this segment is susceptible to many diseases-epithelial cell infection with viruses and Mycoplasma, bronchoconstriction in asthmatic syndromes, bronchitis, bronchiectasis, colonization with bacteria, irritation from noxious gases, and lung cancer. The respiratory mucosa is coated with viscous fluid which is secreted by bronchial glands, globlet cells, and probably by Clara cells* in the distal airways. Some is derived from the intravascular space by diffusion through the blood-air barrier. Special proteins, such as S-IgA and SC, can be added locally along airways by immunoglobulin-secreting plasma cells and epithelial cells. The result is a mucosal fluid layer of great complexity, still incompletely characterized, covering the airway. Few attempts have been made in humans to retrieve selectively mucosal secretion from the trachea and along the bronchi for analysis. However, the preliminary results indicate that these portions of the mucosa actively produce IgA and oi-antichymotrypsin,24 secrete potassium ion (but other electrolytes are lower in proportion than present in plasma),25 and have a low pH of about 6.8-6.9.25, 6 Beating cilia arising from the epithelial lining cells propel these secretions up the respiratory tract; periodic coughing can assist the process. The combination of an in*Clara cells are non-ciliated bronchiolar secretory cells found in the terminal bronchioles. Their function and secretory products have not yet been elucidated in humans. For more information, see Gail D.B. and Lenfant C.J.M.: Cells of the lungs. Amer. Rev. Respir. Dis. 127:366-387, 1983. 18

tact mucosal lining and overlying mucus secretions provides a protective layer which can entrap inhaled particles and repel noxious gases or vapors. Thus, little penetrates or sticks to the respiratory surface, and this seems to be an important function in lung host defense. Bacteria and other infectious agents may transiently colonize the airways, but mucociliary clearance is a dynamic adversary which labors to remove them. Moreover, tight cellular junctions between epithelial cells prevent passage of macromolecules into the submucosa. Several circumstances can alter the intactness of this protective barrier, which may make this portion of the respiratory tract vulnerable or susceptible to disease: (1) nutrition may affect the integrity of mucosal epithelial cells and may allow certain bacteria to adhere; (2) cigarette smoke27 or noxious fumes can disrupt the anatomy of epithelial junctions and enhance passage of airway substances into areas usually inaccessible; and (3) some bacteria can elaborate proteolytic enzymes that may break down IgA and promote their selective colonization.

MICROBIAL FLORA OF NORMAL AIRWAYS A question to be resolved is, “Do bacteria and other microbes inhabit conducting airways in normal lungs?” As mentioned, the mucociliary clearance mechanism is in constant operation and presumably is efficient so that small numbers of bacteria that are inhaled or are aspirated with oropharyngeal secretions should be removed. But are the airways usually sterile, so that when colonization with microorganisms does occur, one can infer that an abnormal situation exists? Several studies28-30 concluded that the bronchi and airways beyond the trachea were sterile in humans without obvious lung disease. In a review of the indigenous microbial flora inhabiting various body surfaces of humans31 the oropharynx was mentioned but the lung was not considered to have flora normal1 2~. The method used may be all-important for the results. To obtain culture swabs of the trachea and lobar bronchi, a rigid bronchoscope (or a 34 French rubber catheter) was passed under local topical anesthesia in a patient. Through it a polyethylene tube (18 French, wrapped with tape near its tip to 19

prevent its direct contact with the bronchoscope) was passed first just beyond the end of the bronchoscope. Then a cotten wool-tipped wire was passed through the polyethylene tube. After swabbing, the tip was retracted into the tube and removed. In ten patients with no evidence of bronchopulmonary disease, no bacteria were isolated from the bronchial swabs; from tracheal specimens, oropharyngeal commensals were present in all, and in four, potential respiratory pathogens were found (Diplococcus pneumoniae, Hemophilus influenzae and coagulase-positive Staphylococcus aweus). What has added confusion to the status of a bronchial flora have been the results of bacterial cultures made from endoscopic washings which were less precisely obtained. For example, only one specimen was sterile in eight control subjects without airways disease when aliquots of bronchial washings, recovered through a rigid bronchoscope, were cultured. All other specimens yielded the same mixture of organisms as found in patients with chronic obstructive pulmonary disease.32 As part of fiberoptic bronchoscopy in normals, bronchoalveolar fluid was cultured, and we noted that virtually all specimens contained multiple oropharyngeal bacteria33; results in normal cigarette smokers were similar to those in nonsmokers. Undoubtedly these “contaminants” collected on the bronchoscope as it was passed through the nose and/or oropharynx into the lungs and did not really represent airway flora. This is so likely that it has been stressed that bronchoscopic washings need not be cultured routinely, for such specimens may be unreliable for patient diagnosis.34 More selective collection methods have been advocated, such as transtracheal aspiration or percutaneous needle sampling of lung infiltrates, to bypass the upper airway flora. Alternatively, better protected catheters that can be advanced through the channel of the fiberoptic bronchoscope seem preferable, because of the popularity and frequent usage of flexible bronchoscopy. In a careful study in 25 normals,36 in which workers used a sterile brush protected within a telescoping double catheter to obtain bronchial samples during fiberoptic bronchoscopy, most cultures from multiple sites in the lower respiratory tract contained bacteria (38 of 52 speci-

mens, or 73%) which were similar to those found in the nasopharynx. Because the colony counts were often so low (O-5 colonies per culture plate), especially when the volunteers were bronchoscoped in a supine or Trendelenburg position rather than a sitting position, the possibility was raised whether these “positive” cultures were always indicative of upper respiratory tract contamination or whether they could denote true lower respiratory tract infection.36 Several conclusions are offered. First, the peripheral bronchi in normal humans are relatively sterile, perhaps completely so, but nasopharyngeal bacteria may be transiently deposited which are then removed by mucociliary or cough mechanisms so that bacterial persistence or colonization is not usual. Second, present sampling devices for use with fiberoptic bronchoscopy are not perfected. We have only discussed bacterial cultures and have not mentioned viruses or other agents such as Pneumocystis carinii and Mycoplasma. Viruses are not present in normal lung secretions and, if cultured, should be considered to represent infection. Some organisms can be inhaled and deposited directly on the alveolar surfaces (with aerodynamic dimensions of 0.5-3 p. in diameter) and not be cultured from the airways, so a dichotomy might be found between airway brushing specimens and cultures of bronchoalveolar lavage fluid. Finally, it is probable that some microbes may exist selectively in the alveoli, perhaps residing in macrophages or in alveolar lining cells, so that the alveolar milieu might have a discernible flora. This will be discussed in the section on infection in immunocompromised hosts. BACTERIAL ADHERENCE TO RESPIRATORY TRACT MUCOSA-A DYNAMIC INTERACTION LEADING TO COLONIZATION Why and how bacteria stick particularly to human mucosal tissues is fascinating and undoubtedly important for the symbiotic relationship that humans develop with them in the gastrointestinal tract, naso-oropharynx, and, to a less important extent, on skin and hair surfaces.312 37, 38 Acquisition of nasal, mouth, and gingival microbial flora is normal, and a variety of aerobic and anaerobic bacteria in21

habit the naso-oropharynx of healthy humans. However, the prevalence of gram-negative bacillary rods, such as Enterobacteriaceae and Pseudomonas species, is 10w.~’ With virtually any alteration in health status,40Y 41 presence of alcoholism or diabetes, or dependent living in a health care rods in the nose facility,42 an increase of gram-negative and throat occurs. In hospitalized patients, increased carriage of these bacteria enhances the risk of nosocomial respiratory infection. Twelve percent of patients in an intensive care unit who became colonized in the oropharynx with a potentially pathogenic bacterium developed pneumonia, usually with the same microbial species. Aspiration of oropharyngeal secretions was the most likely mechanism. Gram-negative rods will adhere to squamous buccal cells (assessed with an in vitro test), and following major surgery, adherence did increase which was correlated with colonization.43 Adherence of bacteria can be assessed also using ciliated epithelial cells obtained from the nose and trachea of humans.44 We measured adherence of Pseudomonas aeruginosa to ciliated cells (from nose and trachea) and compared this with adherence to squamous cells from buccal mucosa. Cell samples were collected from 16 noncolonized individuals undergoing either elective surgery or volunteer bronchoscopy. Adherence (mean ? SEMI to tracheal cells (4.6 -+ 0.8 bacteria per cell) and to nasal cells (4.7 -C 0.6 bacteria per cell) was similar. These values significantly exceeded buccal cell adherence (0.9 t 0.2 bacteria per cell). Because cells from ciliated surfaces bind more bacteria than cells from squamous surfaces, bacterial adherence at these respiratory sites may involve different mechanisms. The enhanced bacterial attachment to ciliated cells may assume pathogenic importance when mucociliary function is impaired. In patients with chronic respiratory disease and a permanent tracheostomy, colonization of the buccal mucosa and of the tracheal area can be independent, and the lower respiratory site is possibly a more accurate indicator of potential lung infection.45 Mechanisms by which bacteria actively attach to airway mucosal cells are multiple. Bacteria can have surface projections (pili) that promote adherence46 or can secrete var22

ious proteolytic enzymes that can impair the host’s mucosal covering. Several very common bacteria47’ 48 produce an IgA protease that can cleave S-IgAl. Salivary protease may be a factor as we1L4’ This may allow these bacteria to colonize more easily. Certainly patients with chronic bronchitis have an increased carriage of Hemophilus and pneumococci in sputum specimens. What effect overall nutrition may have in helping the host resist pathogenic bacteria and retard colonization is unclear. We did note that improving the nutrition in a group of patients with chronic tracheostomies resulted in less Pseudomonas cell binding to tracheal epithelial cells, suggesting a relationship with better caloric intake and nutritional status.50 CHRONIC BRONCHITIS-CERTAIN SECRETORY IGA

BACTERIA

MAY

HARM

Chronic bronchitis is a clinical condition characterized by cough and excessive secretion of mucus in the tracheobronchial tree. It is not due to other specific diseases such as bronchiectasis, asthma, or tuberculosis. The term “chronic bronchitis” is often used loosely, but by definition it should be applied to patients who have coughed up sputum on most days during at least 3 consecutive months for more than 2 successive years.51’ 52 Emphysema often complicates the clinical presentation. Although the precise diagnosis of emphysema is a morbid and anatomical one, its coexistence is usually inferred. Causes of chronic bronchitis have not been elucidated completely but three factors seem to be of particular importance: cigarette smoking, atmospheric pollution, and infection. Other causative factors such as a history of frequent childhood respiratory infections, familial predisposition to respiratory disease, environmental and industrial exposure to air pollutants, or related diseases which impair mucociliary transport in the lung may be important in susceptible people. Cigarette smoking is a significant airway irritant for most patients. An important issue in chronic bronchitis is the role of microbial infection.53-57 Infection does not appear to initiate the disease, but it is probably important in perpetuating the illness and is responsible for the characteristic 23

acute exacerbations. As was just concluded, normal bronchi are almost always sterile. In contrast, pathogenic bacteria can be cultured from bronchial washings of some 82% of chronic bronchitics,“* 32 but many of these organisms may represent oral flora if the specimen is not a transtracheal aspirate or was not obtained with a rigorously protected catheter technique. Routine sputum cultures from patients with chronic bronchitis commonly contain nonencapsulated H. influenzae, Streptococcus pneumoniae, and other oropharyngeal commensal flora. In most clinical series, one or both of these species are recovered from approximately 30%-50% of the sputum specimens and can be considered the baseline microbial flora of many patients with chronic bronchitis. Anaerobic bacteria are infrequent inhabitants and may be retgvered in only 17% of transtracheal aspirate specimens. Sputum carriage of H. influenzae and pneumococci, however, is of uncertain significance. These bacteria tend to persist in sputum during quiescent intervals, and the frequency of recovery is not greatly increased during acute infectious episodes. Development of purulent sputum is not correlated specifically with the presence of one or the other of these bacteria in quantitative cultures. Mycoplasma pneumoniae does not seem to be of real importance, and some studies attribute only l%-10% of acute infections to this organism.57 Other bacteria, such as hemolytic streptococci, S. aureus, and enteric gram-negative bacilli are infrequent causes of acute bronchitis. However, viruses may be a more frequent cause of acute infection than is suspected. About 25% of acute exacerbations were related to these agents and included this spectrum: influenza, parainfluenza, respiratory syncytial virus, rhinovirus, and coronavirus.57 Viral infections are seasonal and usually occur more frequently in winter. It is tempting to speculate that there may be an explanation for the choice of bacteria that colonize the airways of subjects with chronic bronchitis. Special properties of these bacteria and some damage to the host’s mucosal protective layer are part of the explanation. As mentioned, S-IgA accounts for about 10% of total pro24

tein measured in nasal wash fluid and for about 5% of that found in parotid and bronchoalveolar lavage fluid. IgA exists in two molecular forms, designated IgAi and IgAs. The 01 heavy chains are slightly different because of unusual light chain attachment without a disulfide bond to the respective heavy chain. The proportion of IgAiiIgAz is about 60140 in one external secretion, colostrum. Little is known about the values in various portions of the respiratory tract. Both species have been identified in bronchoalveolar lavage fluid. ’ Documenting the immunoglobulin’s precise function is difficult, however, despite the widely held assumption that it is important in protecting external mucosal surfaces throughout the body.11-13 S-IgA has been demonstrated to have antibody activity against certain viruses, bacteria, and common allergens, especially in the upper respiratory tract. Antibodies can be induced either by local respiratory immunization, as discussed, or by natural infection or exposure. Once immunity is established and S-IgA antibody had been produced, protection against homologous microbial challenge, especially with viral agents, can be shown. Because S-IgA has less efficient opsonizing potential than other immunoglobulins and because its interaction with the alternative complement pathway system is not well established, it may be less important in the peripheral airways and alveoli than in the large airways and nasopharynx, where it is an important constituent of mucosal secretions. Although the S-IgA molecule appears to have intrinsic resistance to proteolytic degradation (possibly conferred by its unique glycoprotein moiety SC, which gives a favorable tertiary molecular configuration), it seems that an increasing list of common pathogenic bacteria can elaborate IgA proteases that destroy it. Mulks and colleagues examined 36 strains of S. pneumoniae, 62 strains of H. influenzae, six hospital-acquired respiratory pathogens, and a strain of S. pyogenes for production of IgA protease, a bacterial enzyme whose only known substrate is human IgA.47 IgA protease was produced by 100% of the isolates of S. pneumoniae and 98% of the isolates of H. infhenzae. The enzyme from both species cleaved human serum and S-IgAi proteins, but not human 25

IgA2, IgG, or human serum albumin. None of the hospitalacquired pathogens, such as S. aureus, Klebsiella pneumoniae, and P. aeruginosa had detectable IgA protease activity, a finding indicating that the production of this enzyme distinguishes S. pneumoniae and H. influenzae from the opportunistic respiratory pathogens. Other evidence suggests that P. aeruginosa also produces a protease. Pseudomonas protease cleaves IgGG1 and has activity against S-IgA as we11.62 In another study, Kilian and co-workers sought the actual cleavage points produced by bacteria-derived IgA proteases of the IgA molecule.48 Neisseria meningitidis and S. sanguis were examined, as well as H. influenzae and S. pneumoniae. In addition to protease activity, S. pneumoniae releases an exo- and endoglycosidase that removes a considerable portion of the carbohydrate side chains of IgA, which may impair the immunoglobulin even more. IgA proteases from the above bacteria disrupt the IgAi cx chain in its hinge region, but at slightly different sites; proteases from S. sanguis and S. pneumoniae cleave the Pro (227) Thr (228) numbered amino acids’ bond within the hinge region, whereas H. influenzae-derived enzyme cleaves a ProSer bond at slightly different amino acid positions in the same region. The proteases split only the a1 chain of the SIgA molecule; they had no effect on light chains or on SC and J chain. The metal chelator EDTA inhibited proteases derived from the two Streptococcus species, but not the Hemophilus species. Thus, data demonstrate the existence of at least two types of extracellular bacterial proteases specific for IgAi. Forms of Neisseria (N. catarrhalis and N. meningitidis) can also colonize the respiratory mucosa and oropharynx. Although these are not the principal pathogens in the patient with chronic bronchitis, they illustrate other means of attachment to cell surfaces (e.g., by pili or fimbriae) that promote colonization. Isolates of N. meningitidis from the nasopharynx of carriers and from patients with meningococcal diseases were found to be heavily piliated. Isogenic piliated and nonpiliated meningococcal clones were derived from blood and cerebrospinal fluid isolates in these patients.46 Meningococci with pili consistently attached to hu26

man nasopharyngeal cells in greater numbers than meningococci without pili. Meningococci treated with trypsin or mechanical shearing forces lost pili and exhibited decreased attachment. Attachment of piliated meningococci differed markedly among epithelial cells from different sites. In contrast, nonpiliated meningococci attached equally but in low numbers to all cell types. These data suggest that pili are important mediators of meningococcal attachment. Since the degree of piliation of the Neisseria organisms determines the avidity of attachment to buccal mucosal cells, these bacteria may ensure their successful colonization of the nasopharynx by secreting an IgA protease and by developing pili that can enhance attachment and increase virulence. Therefore, the potential for a bacterium to use its proteolytic enzyme, which can in part split and disrupt an important protective mucosal protein, can give selective advantage to certain bacteria that commonly colonize the airways. That specific protease enzymes have been detected only in S. pneumoniae and S. sanguis, H. influenzae, and two pathogenic strains of Neisseria-N. meningitidis and N. gonorrhoeae-raises the possibility that these bacteria frequently inhabit the oropharynx because they can successfully inactivate some of the mucosal IgA. Chronic bronchitis happens to be an example of a lung disease in which the airways of many patients are persistently colonized with Hemophilus and Pneumococcus. A relation between two independent phenomena does not mean a cause-andeffect relationship exists or that an immunopathogenic mechanism for colonization and infection is proved. Quantification of the two IgA subclasses in airway lining fluid or sputum has not been done in subjects with bronchitis who are colonized or infected with an IgA protease-secreting bacterium, so whether this mechanism is only speculation is untested. A method used by Stockly and colleagues63’ 64 to judge for the presence of infection based on immunoanalysis of 7s and 11s IgA in sputum might be adapted. In this report the relative availability of free SC seemed to determine the molecular form of IgA-i.e., with infection, 7S IgA was increased in sputum and SC was all utilized. Chronic bronchitis is a more complicated disease than one 27

mechanism can explain, but the possibility that an acquired, partial deficiency in an immunoglobulin class can contribute to a common infectious complication is intriguing. CILIARY CLEARANCE While still thinking about the nasopharynx and larger airways, let us describe in more detail the ciliated epithelium which lines the airways from the nose to respiratory bronchioles and provides the moving parts for the mucociliary apparatus. The most striking cell in the mucosa is the ciliated epithelial cell containing a tuft of up to 200 cilia which are remarkable organelles that beat at a fantastic rate and rhythmically propel mucus admixed with other proteins and cellular debris up the respiratory tract. These cells form the uppermost layer of the mucosa. The ultrastructure of normal cilia from nasal mucosa has been described and illustrated in detail.65 When human cilia, located at four levels in the respiratory tract, were studied in vitro to compare the frequency of beating, the frequencies of the nasal, tracheal, and lower lobe bronchial beats were not significantly different, but subsegmental airway ciliary beating was slower than each of the others.66 It seems incredible that respiratory cilia can beat more than 14 times per second or more than 800 times per minute; the rate was somewhat less (600 times per minute) in subsegmental airways, which were about 1 mm in diameter and conservatively estimated to be from a tenth generation branchin of the respiratory tree. Nasal cilia from other studies65T 6F have been found to beat at 12.5 Hz (Hz = beats per second) and 15.2 Hz. Presumably these rates reflect actual in vivo ones, although it is possible that removing the ciliated cells from the epithelial substratum and lamina propria disrupts humoral or autonomic nervous control that regulates the rate and wave form of the beating. Transmission electron microscopy has been indispensable for studying the intrinsic structure of cilia. Cross-sectional profiles (Fig 2) and longitudinal views of cilia can illustrate clearly the anatomy of this minute structure. 28

peripheral doublet

central microtubules

inner

I

spoke

I

outer

nextn dynein

dynein

arm

link arm

Fig 2.-Cross section of a cilium or of the central The assembly of the nine outer microtublar doublets tubules is held together by three kinds of connections: nexin links, and the spokes.

portion of a sperm tail. and two central microthe dynein arms, the

From healthy normals, including nonsmokers, atopic nonsmokers, and asymptomatic smokers, nasal cilia usually have normal ultrastructure (96%), and very few were atypical (4%). Among the atypical forms there were multiple cilia (two or more axonemes* enclosed in one cell membrane), those with central microtubule defects or some with peripheral microtubule defects (the most frequent alteration accounting for 2.6% of “normal” cilia). Of interest, patients with rhinitis and cystic fibrosis had normal cilia and the same proportion of atypical cilia as other normal groups.65 Ciliary Dysfunction Considerable interest structural abnormalities *An

axoneme

is the

has focused on patients who have in the microtubular structure of

core structure

of a cilium. 29

cilia and experience a variety of respiratory and ear infections (otitis media, sinusitis, and bronchiectasis). These defects produce altered or absent motility and represent a fascinating linkage between abnormal airway host defense and a propensity for recurrent or chronic infection at multiple sites along the respiratory tract. Among the primary ciliary dyskinesia disorders, very specific lesions have been noted for several syndromes. The original abnormality, absence of dynein arms, which causes incoordination of cilia, has been implicated as the structural defect of Kartagener’s syndrome.68Y fig An absence of radial spokes that connect the peripherally located doublets of microtubules with the central doublet was the second defect recognized.70 This disease, while causing the usual features of sinusitis and bronchiectasis, is not associated invariably with malrotation of viscera, i.e., situs inversus, nor does it occur only in males. Next, cilia were found to have a deletion of the central tubules and the apparent adaptation of transposing one of the7peripherally located doublets to the center of the axoneme. The rate of tracheal ciliary transport is severely impaired, but some activity is retained. More sensitive assays have found that the dynein armdeficient cilia, thought to be nonmotile, actually have some movement. This, plus the fact that the other major types of structural defects only slow and alter cilia wave form motion but do not produce nonmotile forms, has prompted7’ renaming the congenital “immotile ciliary” syndromes as diseases of “ciliary dyskinesis.” However, it is difficult to erase the immotile concept, and this name may persist. Although this classification is based on very specific lesions, minor defects or more generalized multiple alterations are being recognized. As noted already, normal people may have a few percentage of structurally atypical cilia. Defects in cilia can be acquired with certain infections or aerosolized chemical toxins,73 or the “immotile” cilia syndrome of the Kartagener’s variety (with absent dynein arms) can be found in patients with only chronic bronchitis and bronchiectasis.73 In a group of native patients with bronchiectasis,74 bronchial or nasal cilia all lacked dynein arms, but cilia also contained a variety of other abnormalities including missing tubules, extra tubules, and mis30

placed tubules. Also observed were compound cilia, intracytoplasmic cilia, vesiculated cilia, and cilia with long, winglike folds. In all of these patients, pulmonary mucociliary transport rates were either absent or markedly reduced. The authors suggested that these abnormalities were due to a mutation or group of mutations which was the underlying cause of their bronchiectasis. Another patient series75 included 17 children with immotile cilia syndrome due to various defects in the assembly of dynein arms in the cilia. This ultrastructural analysis revealed cilia to lack either inner arms or outer arms, or both. The authors suggested that the spectrum of defects contributing to dynein-deficient cilia reflects separate genetic determinants, which is further evidence that the immotile cilia syndrome is genetically heterogeneous. Despite quite specific ultrastructural differences in cilia, no significant differences were evident in the clinical course of the respiratory disease in affected subjects, or in the incidence of situs inversus that affected 50% of subjects. To summarize, patients who have recurrent or chronic respiratory infections can acquire nonspecific changes in the morphology of cilia which could complicate the identification of genetic defects. However, these nonspecific changes are different from the specific abnormalities of the dynein arm and radial spoke. It is important that an adequate number of biopsy specimens and sections be viewed to define accurately an ultrastructual defect. Chao et al. examined biopsy specimens from the nose and the bronchial areas, and for quantification purposes at least 50 cross sections of cilia from both biopsy sites were inspected.75 The dynein arm defects are the most common abnormality noted among the three ultrastructural categories, and the dynein defects are genetically heterogeneous. A recent study65 addresses quantification of ciliary defects in normals and in patients with various ciliary dyskinesia disorders. This gives some numbers for what has been evolving in the literature about ciliary diseases-that some of the defects are not so obvious and may overlap with or require separation from similar but nongenetic forms of chronic respiratory infection. Because the spectrum includes subtle forms, the diseases may go unrecog31

nized and actually may be more prevalent than is suspetted. Rossman and colleagues65 used nasal mucosal brushing or curettage to obtain cilia samples for ultrastructual analysis and for ciliary beat frequency or wave form beating characteristics. As already mentioned, various groups of normals and subjects with rhinitis or cystic fibrosis had normal beat frequencies of approximately 12.5-15 Hz. In subjects with a dynein defect, a radial spoke defect, and a translocation defect of cilia, mean beat frequencies were reduced to 6, 9.6, and 10 Hz, respectively. Whereas normals have only a few percentage of atypical cilia, in the dynein defect group 96% of all cilia were missing dynein arms, whereas in the spoke and translocation defect groups, 72% and 32%, respectively, of cilia were abnormal. Thus, in the latter groups normal cilia were observed in many instances, pointing to the necessity of viewing many cilia before concluding a diagnosis. With the specific defects, characteristic waves of motion were noted, described as rotational and vibrational (for the 40% or so remaining motile cilia found) when a dynein deficiency existed, corkscrew-like if radial spokes were missing, and grabbing when a translocation defect existed. Clearly, considerable expertise must be assembled to perform the structural and kinetic analyses just outlined and to subdivide correctly the clinical abnormalities, but such seems to be the state of the art as the diagnoses become more quantitative and more functional. Ciliotoxic Infections: Viruses and Mycoplasma pneumoniae Malfunction of ciliary transport may result from intrinsically defective cilia, as discussed, and from infections that are ciliolytic (My. pneumoniae) or actually destroy the cilia-containing epithelial cells (viral agents). In a subtle way, viral infections can be associated with prolonged airway dysfunction by causing peripheral small airway obstruction and hyperreactivity. Mild influenza A infection in the upper airways of young, ostensibly healthy college students was found to cause increased airway resistance and hyperreactivity which persisted for almost 2 months, well beyond the duration of clinical illness.76 32

Whereas the antiviral agent amantadine appeared to arrest virus proliferation and presumably the associated inflammatory response in peripheral airways, it did not prevent the development of bronchial hyperreactivity. Viruses, especially myxoviruses, which include influenza, infect and secondarily destroy ciliated epithelial cells along the conducting airways; this infection can lead to denuded areas without functionally ciliated cells. Coughing, a prominent symptom of the disease, can be viewed as a compensating host defense mechanism to clear secretions that are ordinarily swept out of the bronchi and trachea by ciliary motion. Although certain viruses can infect mucosal epithelial cells and thereby affect ciliary function, the greatest impact of viral infection may be on the lower respiratory tract. A viral infection can doubly jeopardize the respiratory tract by causing a primary cytotoxic effect of airway lining cells and by predisposing to pneumonia. Invading viral droplets or infected epithelial cells that are shed from the mucosa can also reach the alveoli (aspiration?) and infect macrophages. As a consequence, these viral-infected phagocytes lose some of their bactericidal effectiveness.77 Thus, bacterial superinfection with S. aureus, for example, is not an infrequent sequela of viral respiratory infection. These bacteria may more easily penetrate the denuded areas of bronchial mucosa or may not be killed readily by alveolar macrophages. Mycoplasma can cause a similar problem for the ciliated epithelial cells in the conducting airways. Mycoplasma organisms seem to be ciliolytic as well as ciliotoxic, causing ciliostasis. Of the three species of mycoplasmas that are pathogens in the human host, M. pneumoniae is the one that affects the lungs. Its natural target cells are the epithelial lining cells of the conducting airways. With tracheal organ cultures from hamsters and human fetuses, Collier and co-workers78-81 noted that Mycoplasma caused ciliostasis and cytopathologic changes in cilia. The organism has a specialized terminal structure or tip by which it attaches to epithelial cells and cilia. Mycoplasmas do not invade the cells but remain on the surface. Either a mycoplasma surface protein or attachment to sialic acid moie33

ties mediate adherence between the special tip and host cells. The binding site seems to be a protein receptor, for pretreatment of cells with trypsin can remove it. The attachment of mycoplasma is modulated somewhat by availability of S-IgA antibody in the host, so the immune status of the airways is important.” Once infection has occurred. antimycoplasmal antibody in sputum in other immunoglobulin classes can be identified (IgM and IgG), attesting to the systemic as well as local immune response this organism can elicit. As with viral infections, persistent cough is a feature of mycoplasma respiratory infection and in fact may be a necessary host compensation for poor ciliary clearance. Bordetella pertussis also attaches to ciliated mucosa and is even more particular than Mycoplasma about adhering just to cilia and not to nonciliated epithelial cells or squamous cellss3 Bordetella pertussis localized to the proximal portion of the ciliary shaft. The cell membranes of Bordetella and the cilium did not fuse and a glycocalyx layer filled in between. Although this bacterium is of interest because of its characteristic attachment to cilia, whooping cough is no longer a serious disease threat, because of antipertussis vaccination. However, it is unclear why this vaccine works. Usually, parenteral immunization does not elicit any local mucosal antibody that would prevent colonization or replication of the organism, so serum antibody must diffuse through the mucosa in some way. CYSTIC

FIBROSIS

Cystic fibrosis is a prevalent genetic disease in the American Caucasian population (autosomal recessive gene carried by approximately one in 20 people), is still of unknown cause, and involves many organ systems. However, respiratory disease usually becomes the most significant problem for these people, and chronic infection leading to respiratory insufficiency may kill the patient prematurely.84-s6 Cystic fibrosis involves several components of respiratory host defense-mucociliary clearance,87’ 88 cough, local immunoglobulins, and phagocytic cells. The disease is not just a tragedy of infancy, affecting one in 1,600-2,500 live-born 34

infants and young children, but increasingly is a disease of teenagers and g oung adults, and is being recognized in older adults.“-’ Moreover, it is a disease that has received enormous research effort, but as yet the basic biochemical abnormality remains undetected. Recent work elucidatin ion transport across fibroblastsg6 and epithelial cellsg7’ ’ 5 seems to be focusing on relevant cellular mechanisms that may give a clue to the underlying lesion. At birth, cystic fibrosis subjects supposedly have normal lungs, so a postnatal sequence of respiratory infection and injury must occur; however, the precise order of events or the interaction between them remains uncertain. Possibly a viral respiratory infection initiates a bronchiolitis, which in turn stimulates excessive mucus and respiratory secretion in the peripheral airways; clearance mechanisms may be faulty (poor coughing or ciliary action), although overt defects in the ultrastructure of epithelial ciliary cells have for the intrinsic ciliary not been found,65 as demonstrated diseases. The combination of excessive and stagnant secretions which are retained in the airways causes airway obstruction and provides a good nutrient medium for microbes entering the respiratory tract. Alternatively, replicating microbes or their extracellular enzymes could cause mucosal irritation that stimulates goblet and bronchial gland secretions or could produce direct injury to cilia with ciliotoxic substances. Whatever comes first, the collection of inflammatory cells (PMNs predominantly), proteolytic enzymes (elastases),” toxic oxygen radicals or halide ions, and bacterial or viral substances plus mucus and sloughed cellular debris creates a purulent, tenacious, and biologically destructive exudate that can accumulate in the airways, ‘O”, lo1 S. aureus has been accorded an important role, because this species of bacterium often follows significant viral respiratory infections and it possesses an array of cellular enzymes that can cause many of the destructive processes described. Pseudomonas aeruginosa infections, usually the bane of cystic fibrosis subjects nowadays, have only in more recent ears become so important and universal in cystic fibrosis.’ t7‘, lo3 The reason for this microbial change is uncertain but in part may reflect the efficacy of antistaphylococcal antibiotics used in cystic fibrosis subjects, often 35

in a prophylactic way to suppress infection. Moreover, the conducive milieu in the airways of cystic fibrosis patients promotes colonization with other microorganisms that can lead to infections with mucoid Escherichia coZi,104 Pseudospecies. At present, it monas cepacia, and Aspergillus seems that currently available antibiotics with potent activity against P. aeruginosa, i.e., aminoglycosides and carbenicillin derivatives, may be controlling this bacterial species better, but at the expense of creating other equally potent infections. The net result in the lung seems the same, however: widespread bronchiectasis, recurrent bronchitis, and pneumonitis that culminate in respiratory insufficiency. Previous investigation of cellular and humoral immune components of lung defenses in subjects with cystic fibrosis found these to function normally, with one major exception, whole serum appears to interfere with the phagocytic function of alveolar macrophages. There no longer seems to be support for an earlier finding that the serum of cystic fibrosis patients could inhibit ciliary beating now that an allhuman assay has been used.67 Whole serum from patients homozygous for cystic fibrosis inhibits the ability of rabbit and human alveolar macrophages to ingest and destroy Pseudomonas bacteria.106-10g This effect has been variously attributed to a serum deficiency, to a heat-labile factor, or, more recently, to a heat-stable inhibitor factor in cystic fibrosis sera. “Bactericidal-blocking” antibodies have been suggested as an explanation for the selective inability of whole serum from cystic fibrosis patients to support Pseudomonas bactericidal activity. This abnormality seems to have its major impact on the respiratory tract, since systemic or extrapulmonary infections are rare in cystic fibrosis, even in patients with severe pneumonia who should have an increased incidence of bacteremia and sepsis. Serum agglutinating titers to Pseudomonas antigens and antibodies to other microorganisms are generally quite high in cystic fibrosis subjects, indicating adequate and appropriate primary and secondary (booster) humoral responsiveness. In fact, an excessive level of IgG has been noted in cystic fibrosis patients with more severe lung disease, whereas those with relative hypogammaglobulinemia had 36

complex deposition in less. Ilo The possibility of immune lung tissue as an explanation is an attractive mechanism that might increase inflammation. Because of the specific nature of this “humoral” defect in the respiratory tract, one can rationalize what the defect(s) might be. Optimal phagocytosis of Pseudomonas by PMNs or alveolar macrophages requires the presence of heat-stable opsonins111-113; of the heat-stable opsonins, IgG has superior protective activity when compared with S-IgA or IgM. Similarly, IgG antibodies in convalescent sera have been shown to augment phagocytosis of Pseudomonas organisms f;en in the absence of heat-labile (complement) Because of the importance of immune IgG in opsonins. the phagocytic process, and because of special constraints in antibody binding to macrophages imposed by specific cell surface receptors, it is likely that such an opsonic antibody deficiency would be of the IgG class.i14, ‘15 While cystic fibrosis patients have a generous antibody response in serum, sputum, nasal secretions,i16 and, to a lesser degree, in lung lavage fluid to P. aeruginosa somatic antigens, the functional capacity of these specifically purified antibodies has not been studied directly. Several years ago, my colleague, Dr. Robert B. Fick Jr., and I began to examine the structure and function of antiPseudomonas IgG opsonins in serum and respiratory secretions of subjects with cystic fibrosis and in patients with naturally acquired Pseudomonas lung infections. To extract pure antibody specimens from sera and secretions, a preliminary protein fractionation was done to concentrate various immunoglobulin classes, which was then followed by affinity chromatography against a specific P, aeruginosa lipopolysaccharide from a mucoid strain.‘17 This produced “pure” IgG high-titered antibody suitable for immunochemical studies. When IgG antibody from cystic fibrosis serum was compared with similarly prepared IgG from normal subjects immunized with a lipopolysaccharide Pseudomonas vaccine, the cystic fibrosis opsonins were less effective in promoting phagocytic uptake and killing by normal alveolar macrophages.‘13, ‘18 This seems to be caused by poor cellular attachment of the cystic fibrosis IgG antibody, possibly due to a functional abnormality in the Fc portion 37

of the IgG molecule. In respiratory secretions from cystic fibrosis patients, some IgG antibodies were found to be fragmented and to have lost the Fc portion of the heavy chains, resulting in Fab kind of fragments. This heightened susceptibility for IgG to be cleaved has been traced to proteolytic conditions that exist in the secretions of cystic fibrosis subjects because of high elastolytic activity. Such activity is caused by PMN elastase and by a P. aeruginosaderived protease.“, lo1 The overall result in the lungs could be the formation of IgG fragments that could act as cytophilic antibody or blocking antibody when attached to the surface of a phagocyte such as a macrophage or PMN. This could interfere with the usual receptor binding of other IgG opsonic antibody and reduce opsonin-mediated phagocytic uptake of bacteria. These mechanisms are under investigation. Preliminary evidence on the distribution of IgG subclasses in lung secretions from cystic fibrosis subjects shows a high proportion of IgGi and IgGs (accounting for about 80% of the IgG subclass proteins) and minimal amounts of IgGa and IgG4.11’ Presumably, much of the specific anti-Pseudomonas lipopolysaccaride antibody resides in the IgGi and IgGs subclasses. In summary, it appears that the functional capacity of IgG opsonin is diminished because of poor Fc attachment to alveolar macrophages, which could be an intrinsic defect in the antibody molecule. This is compounded in the inflamed and Pseudomonas-infected airways, where a bacterial protease is present that can degrade IgG. In effect, this may create more defective antibody. Agglutination and coating function may be preserved, but ineffective attachment to appropriate phagocytes may be lacking. IGG

DYSFUNCTION

Among the respiratory mucosal proteins of the nasopharynx and conducting airways, the concentration of IgG is much less than IgA and accounts for only 1% or so of the total protein. However, in the alveolar area, IgG is the major immuno lobulin and may represent as much as 10% of the protein!39 120-122 Although IgG is supplied to the airways by several mechanisms, diffusion from the intravas38

cular space is the major source in normals.lz3, 12* IgG antibody is intimately involved with phagocytic cells and complement factors because of its superior opsonizing function and specific receptor attachment to cell membrane surfaces. Thus, the impact of IgG dysfunction could be presented later in this review with the discussion of abnormal host factors in the alveolar milieu that predispose to infection. Abnormalities of IgG are coupled with IgA deficiency and cause diseases characterized by upper respiratory tract infections (sinusitis and otitis media) and bronchiectasis. Also, IgG is associated with airway hypersensitivity (asthma and certain organic dust inhalation diseases), so it is appropriate to include IgG-related problems in the present section on the conducting airways. Whereas inactivation of IgA by bacterial proteases is an attractive hypothesis pertaining to bacterial colonization and perhaps to infection in patients with chronic bronchitis, abnormalities of IgG are clearly associated with infection. Hypogammaglobulinemia is a well-established cause of infection, and common variable immunodeficienc is one of ?:5, lz6 The the most frequent forms of immunodeficiency.’ hallmark is a decreased concentration of some or all of the serum immunoglobulins, especially IgG. Antibody responses and, in some patients, cell-mediated immune responses can be diminished, too. A variety of autoimmune phenomena can be detected in these patients, including overt autoimmune diseases. The disease is not strictly familial or restricted to youth, like X-linked infantile agammaglobulinemia, and it is peculiar in that its onset can be (1) late in life, occuring in the fourth to seventh decades, (2) apparently abrupt, and (3) intermittent or waxing, with spontaneous remissions. The disease can be acquired, especially in the adult.lz7 Respiratory tract infections (pneumonia, sinusitis, and otitis media) and gastrointestinal infestation with Giardia lamblia are usual manifestations of disease. Bacterial infections with encapsulated organisms that require opsonizing antibody (IgG) for efficient host phagocytosis are commonl’* A selective deficiency of IgA12gP131 or of SC!z’ can be associated with susceptibility to infections. However, possible deficiencies involving IgA and IgG132, 133 or selective IgG 39

subclasses134-13” are complex and often subtle. In particular, abnormalities in IgG subclasses have awakened interest in the importance of these individual types for lun disease deficiencies associated with respiratory infections’ 8 7-13g and elevations of IgG4 associated with asthma140-144 and hypersensitivity lung disease.145’ 14’ To document the IgG-IgA deficiency relationship, serum Sam3 les were analyzed for IgG subclasses Gi, Gz, Gs, and G 4 ’ from 37 patients with selective IgA deficiency and associated diseases such as recurrent infections, autoimmune disorders, malabsorption syndromes, and allergic diseases, and from 11 healthy adults with an incidental finding of selective IgA deficiency. In seven of 37 patients in the disease group, low (~0.01 gm/L) or unmeasurable serum IgGz values were found; in sera from the 11 healthy IgA-deficient people, IgGz values were normal. In addition, very low levels of IgG4 were found in the patients with combined IgGs and IgA deficiency. IgGi and IgG3 levels tended to be elevated in all of the IgA-deficient sera, suggesting a compensatory increase. The disease correlation that was striking was in sera showing a combined IgA and IgGz (and IgG4 as well) deficiency, for six of seven patients had recurrent upper respiratory tract infections. However, no details were given about the age of the IgA-IgGs-deficient patients, about the causative microorganisms, or about the anatomical site of the infections. Importantly, no samples of airway secretions were analyzed in this report for secretory IgA or for IgG subclasses. In addition, Oxelius and colleagues133 studied sera from 22 patients with ataxia-telangiectasia, ten of whom had concomitant IgA deficiency, which is a common occurrence in this disease. An imbalanced IgG subclass pattern was noted in all, with IgGz low in all and IgG, undetectable in 19 of 22 patients. Associated chronic respiratory infections correlated best with the IgG deficiencies, and not with the presence or absence of IgA. IgGi was elevated in all subjects. Again, few clinical details were given and no respiratory secretions were analyzed. Beck and Heiner137 found the best evidence of an IgG subclass deficiency alone being associated with recurrent bacterial respiratory infections. Four subjects with severe 40

recurrent sinopulmonary infections and bronchiectasis were found to have virtually absent levels of IgG4 in serum and no striking abnormalities in other IgG subclasses or other immunoglobulins. In contrast, elevated levels of IgG4 are associated with lung hypersensitivity disease. Prior work has implicated IgG4 as a reaginic-type antibody that can trigger mast cell degranulation and mediator release140-‘42; hence it may be a factor in type I immediate hypersensitivity reactions in certain circumstances. When bronchoalveolar lavage fluids from subjects with pigeon-breeders’ hypersensitivity lung were analyzed, they contained increased amounts of hi&h. 145,146 It is possible that this IgG4 represents a specific antibody response in the airway to inhaled avian antigens found in excreta. Clearly, this IgG4 subclass is important in the host defense apparatus of the respiratory tract: its absence may permit bacterial infection to occur, and its increase may be associated with hypersensitivity phenomena. But why its effect is so pronounced is unclear. The IgG subclasses are known to have individual functions that relate to different half-life turnover,1472 14’ very different concentrations in serum,14g-152 probably reflecting the different T l/2, and variable ability to bind complement.153 In lower respiratory secretions, sampled by bronchoalveolar lavage, from normal human cigarette smokers and nonsmokers, we noted’24 that IgGi and IgG2 constituted most of the total IgG (about 91%-93%), as was found in serum (about 97%). IgG3 and IgG4 were detected in small amounts. IgG4 in bronchoalveolar lavage fluid of smokers (mean, 5.0 p.g/ml of lavage) and nonsmokers (mean, 4.0 pg/ml), representing 31 subjects, was increased, compared to IgG4 in their serum; IgG3 levels were variable. However, it seemed as if IgG4 accumulates preferentially in the lower respiratory tract, especially in smokers. As IgG opsonic antibody is an important function of this immunoglobulin, the availability of y chain-specific receptor sites on phagocytic cells allows attachment of IgG antibody. The number or affinity of these cell surface sites are quite different for the four subclasses. Blood monocs&sg6for example, bind IgG, and IgG3, but not IgG2 or IgG4. In the lung, rat alveolar macrophages show avid binding of 41

heterologous IgG subclasses 1 and 3, appreciably less for IgG4 (50% less binding), and no IgG2 binding.157 Well5 examined IgG subclass binding to human alveolar macrophages in a homologous system and found that 30% of macrophages will bind IgG,, 10% bind IgGi, but virtually none bind IgGz and IgG4 (Fig 3). Preliminary evidence reported by Young and colleagues158 indicates that human alveolar macrophages do have membrane-bound or cytophilic IgG immunoglobulin, with most macrophages exhibiting IgGi (about 51% of cells), but an almost equal percentage (45% of cells) have IgG4; cells staining for IgGz and IgG3 are in lower percentages (12% and 4%, respectively). Thus, something of a paradox may exist on the membrane of alveolar macrophages in that IgGi and IgG4 might accumulate on the cell surface, but active binding with another subclass, IgG3, might be required for particle attachment that triggers phagocytic ingestion. Similar evidence has been given to explain macrophage ingestion of certain strains of S. auSMOKERS

and

NONSMOKERS

24HR

BAL

AM

CULTURE

40 .

o nonsmoker A smoker

2 8.

0

.

. JiLIL-

00 -AIgG I

lgG2

IgG 3

Fc RECEPTORS

lgG4

IgM

E

of AM

Fig 3.-The percentages of adherent respiratory cells, in culture for 24 hours, that formed rosettes with various IgG subclass-erythrocyte complexes. IgM EA (coated with IgM that has no cell receptor site, hence a negative control for binding) and erythrocytes (Ej alone are controls. BAL, bronchoalveolar lavage; AM, alveolar macrophages. (From Naegel et al.‘15 Reproduced by permission.) 42

reus containing protein A. The bacteria attach by protein A to surface-bound IgG1.15s-160 Current evidence indicates that IgG4 does not bind particles effectively to alveolar macrophages and hence would not perform well as an opsonic antibody113, ‘14 that could enhance phagocytic uptake of bacteria (encapsulated ones). Since this subclass does not bind complement, it is further limited in mediating complement receptor binding (via C3b on human macrophages) and in creating lytic reactions. However, IgG4 does seem to be a natural cytophilic immunoglobulin. Therefore, it is unclear why IgGz and IgG4 should have such an impact to render the human host susceptible to respiratory infection. CLINICAL PRESENTATION AND EVALUATION OF THE “ADULT" DEFICIENCY SYNDROMES Within the nasopharynx and conducting airways, several parts of the host defense apparatus can be faulty and lead to problems with respiratory infections. Single abnormalities can account for widespread illness, as poorly moving cilia can cause stagnation of mucus secretions in sinuses and the bronchial tree or absent immunoglobulins, such as IgA or certain IgG subclasses, allow bacteria to colonize and infection to develop. A disease like cystic fibrosis may not feature an overt defect, but several problems in aggregate-tenacious mucus, proteolytic destruction of IgG, and mucoid Pseudomonas-stop up the airways, and bronchiectasis, sinusitis, and bronchitis follow. Translating a description of these diseases to a diagnostic situation, however, can be difficult unless the physician remains alert and receptive to the possibility that one of these diseases could exist and knows how to obtain basic laboratory information that would confirm the suspicion. In the preceding sections, a synopsis of the altered pathophysiology or host defect was presented; in this section, we offer an overview of the clinical presentation and outline an approach to differential diagnosis. Management of recurrent respiratory infections is routine for physicians who care for patients with chronic lung diseases. In most instances the patient’s underlying ob43

structive airway disease, chronic bronchitis, associated cigarette habit, or bronchiectasis provides a ready explanation for the infection. Thus, an exacerbation of bronchitis or a complicating bacterial pneumonia following a viral syndrome may be anticipated and is no surprise. Although some of the medical therapies used to manage infections (chronic obstructive lung disease, chronic bronchitis) in the ambulatory patient have not been proved effective with utmost scientific rigor, the general regimen of intermittent use of antibiotics and bronchodilators, promotion of good drainage and flow of secretions, the judicious use of low doses of corticosteroids, and prophylactic immunization (viral and pneumococcal vaccines) usually suffices to control infection in most patients. Occasionally such measures are not adequate for a particular patient. A patient with chronic lung disease may have an abrupt increase in the frequency of respiratory infections or have a series of recurrent bacterial pneumonias, or the physician may be confronted with a relatively young person who seems troubled with an unexpected number of respiratory problems which are inappropriate for age, smoking status, and peer group. These may not be in the form of overt lung infections but rather symptoms of cough and excessive sputum, poorly controlled allergic rhinitis or asthma, frequent sinusitis, and bouts of otitis media. The complex of symptoms may be subtle in aggregate and may not be fully appreciated by the patient who has learned to adjust to or accept them. When should the physician suspect that the patient with chronic lung disease has departed from the usual pattern of response and entered an unexpectedly accelerated phase, or that the younger patient is experiencing more trouble than is normal? The criteria are not established, nor is there a specific number of infections that would be considered excessive. Usually, the physician is left only with his intuition that the patient is not typical or following a usual pattern. Should the usual medical regimen be intensified to break the infection cycle with more vigorous therapy, or should further diagnosis be undertaken to elicit a better explanation or uncover another cause? Whatever the choice of action, two points deserve emphasis: (1) acquired defects 44

in host defense mechanisms can occur late in life and can complicate existing disease, and (21 subtle forms of immune deficiency can exist undetected and be of little consequence at the time but eventually compound health problems later. Respiratory tract infections are the unifying feature of these immunodeficiency syndromes and usually provide the clinical clue to their existence. Such deficiencies are not the overt and severe syndromes that comprise the “pediatric” group of diseases and are usually recognized and diagnosed in early lifelz6, I”; rather, they are mild, subtle, nonlethal forms, perhaps heterozygous in expression, which are becoming increasingly evident in adults. It is important to document the presence of such “adult deficiencies” in affected patients because specific replacement therapy is available in some instances, or more intensive medical care once the problem has been uncovered can improve longterm health prospects. The deficiency syndromes encountered most frequently include cystic fibrosis, immotile cilia disease, and acquired hypogammaglobulinemia. In the following discussion some case material will be injected to introduce each deficiency and to illustrate the kind of diagnostic and management problems the syndromes can create in adult patients.127 CASE 1. CYSTIC FIBROSIS.-A 31-year-old single white woman was referred because of bronchiectasis and recurrent P. aeruginosa pulmonary infections for 3 years. She had had mild asthma in childhood and a presumed bacterial pneumonia at age 4 years; otherwise she was healthy and grew and developed normally. She was quite active in athletics. At age 17 years she experienced the insidious onset of cough productive of purulent sputum. A thorough evaluation, including bronchography, disclosed saccular bronchiectasis involving the proximal bronchi of the right middle and the right lower lung lobes. Her general health remained good and she continued to participate in athletics during college. From age 21 to 27 years she had little difficulty, although she had several acute respiratory infections each year which were easily controlled with short courses of antibiotics. At age 28 years a sputum culture yielded P. aeruginosa, and thereafter the episodes of bronchitis and bronchopneumonia due to this microorganism were more frequent and difficult to treat. Hospitalization for courses of intravenous aminoglycoside anti45

biotic became necessary, and asymptomatic intervals were rarely more than several months long. Maxillary sinusitis occurred. Attempts to categorize the patient’s lung disease were made, but a serum or-antitrypsin level and sweat electrolyte values were reported as normal. Her family had no history of lung or hereditary diseases, and her only sibling, a teenage sister, was healthy. When evaluated, the patient was normal in appearance but about 10% below ideal body weight (53 kg). She had extensive lung involvement with coarse rales throughout both lung fields, mild peripheral cyanosis, and cardiac findings suggesting increased right heart pressure; finger clubbing was absent. Her Shwachman scorelo that is calculated to assess overall health was approximately 60. Pulmonary function tests showed both restrictive and obstructive components to be present. Arterial blood gases (while breathing ambient air) were: Pao2, 56 mm Hg, Paco2, 36 mm Hg; and pH, 7.5.

COMMENT.-Because of the recurrent Pseudomonas respiratory infections and extensive lung disease, this woman appeared to have cystic fibrosis. At this point her clinical picture seemed obvious, yet it was clear in retrospect that just a few years before such a diagnosis would not have been easy to make, for she was ostensibly healthy, except for a few extra yearly respiratory infections and areas of known bronchiectasis in her right lung. Moreover, another physician had suspected cystic fibrosis but the diagnosis was not confirmed, largely because sweat electrolyte values were within normal limits. The onset of Pseudomonas lung infections 11 years after the beginning of the cough and episodes of bronchitis, plus the accelerated deterioration of lung function, focused her problems and made the likelihood of cystic fibrosis more probable. Several additional laboratory indices were helpful in the diagnosis. Numerous sputum cultures were positive for mucoid P. aeruginosa; S. aureus and other pathogenic bacteria were not present. Usual laboratory measurements made on serum and urine were normal. Fecal fat excretion was 6.0 gm/24 hours when the patient was on a diet that included 100 gm of fat per day. The serum carotene level ws 70 pg/lOO ml. Pancreatic exocrine function was not assessed. Results of an oral glucose tolerance test and D-xylose test were normal. Sweat electrolyte levels were obtained, using pilocarpine iontophoresis, on several

occasions. From six determinations, the mean sodium content was 82.0 mEq/Liter (i: 4.0, SEMI. The mean amount of sweat produced per test was 190.0 mg 5 8.0. We have seen several patients similar to this one in whom cystic fibrosis seemed to be a plausible and unifying diagnosis to explain their chronic lung disease. In all, the constellation of health problems did not fit together well until adulthood (ages 18-31 years) to make the diagnosis obvious, although in retrospect each had some clue(s) that should have made one more suspicious. In several, spurious laboratory data on sweat electrolyte values sidetracked physicians pursuing the diagnosis. Cystic fibrosis in older patients seems to affect primarily the respiratory tract, whereas gastrointestinal symptoms and malabsorption which are usual in most patients (85%) or substantial deviations from ideal weight and growth are absent.84, 85 Moreover, these patients do not have an excessive number of illnesses in infancy and childhood that raise suspicions about their health. By the late teens, most have developed a productive cough and have sought medical evaluation for it. Cigarette smoking is a most unusual habit in these people. Confronted, then, with a young nonsmoker who appears to have chronic bronchitis for an unexplained reason, the physician should undertake further diagnostic measures. Individuals with cystic fibrosis can be atopic and have allergic symptoms, too. Several subjects have had seasonal hay fever, rhinitis, and nasal polyps, but none had frank wheezing and an asthma syndrome. Nasal polyps developing in a youngster without atopic illness raises the possibility of cystic fibrosis. Pseudomonas may be isolated from sputum cultures initially, despite the patient’s limited exposure to broad-spectrum antibiotics. A mucoid strain is often a clue that cystic fibrosis may be present, since recovery of this strain even from people with chronic lung diseases is very infrequent. On chest radiographs, the parenchyma usually shows streaky infiltrate and betrays the existence of considerable lung damage. Episodes of aspiration and lung abscess and infections with anaerobic bacteria are infrequent.161 In all of our patients, analysis of sweat composition was 47

used to confirm the clinical impression that these patients had cystic fibrosis. Abnormal sweat composition has been the most reliable laboratory method for the diagnosis of the disease, and the abnormality is present in all affected patients. However, it is thought that this test is less reliable in adults over the age of 20 years, and that sweat sodillm levels in particular may be elevated in older normal subjects and in the parents of children with cystic fibrosis. Davis and colleaguesr6’ reexamined this issue in a large group of adult patients with chronic respiratory diseases that might be confused with cystic fibrosis, using pilocarpine iontophoresis as the sweat stimulus. Of 187 adults (mean age, 54 years -+ 14.7 SD) without cystic fibrosis, seven subjects (< 4%) had a sweat chloride value above 60 mEq/L, a value that would be considered abnormally high or in an overlap range; the clinical history readily excluded the likelihood of cystic fibrosis in these subjects. For the others, including normals, sweat chloride values were in the 21-38 mEq/L range; in contrast, values in cystic fibrosis subjects (mean age, 22.5 years) were about 100 mEq/L. The authors concluded that a properly performed sweat test and analysis did have excellent discrimination for identifying cystic fibrosis subjects, if appropriate clinical features are present, and for excluding this diagnosis in patients with forms of lung disease that potentially could be confused with cystic fibrosis. Therefore, a sweat chloride value above 60 mEq/L is a laboratory abnormality that helps establish the presence of cystic fibrosis if the clinical picture is appropriate. Sweat simulation and analysis induced by iontophoresis is a standardized test and now widely available, but we wish to emphasize that several of our patients had undergone peculiar attempts to create or induce a suitable sweat sample, which may have contributed to erroneous electrolyte values obtained on these tests and hence to a delayed diagnosis. The age at which the diagnosis is made is increasing for cystic fibrosis generally, and many reports have identified older subjects in the third and fourth decades. Within the mild end of the spectrum of disease, cystic fibrosis may account for more cases of chronic respiratory illness than heretofore suspected. Of more importance, most patients 48

with cystic fibrosis are living longer, and the mean age has risen to the later teenage years, with many surviving well into adulthood. The chest physician is quite likely to encounter college-aged and graduate patients with cystic fibrosis, as well as those further along in their professional careers. Their medical care in many instances has not been neglected, but is often fragmented and inconveniently arranged. Having outgrown the comprehensive care of the cystic fibrosis center, and perhaps the university student health facility, they may be uncertain which physician is best equipped to provide them with continued care. The chest physician certainly has an important role because the therapy and control of recurrent respiratory infections, diminishing pulmonary function, hemoptysis, car pulmonale, and respiratory failure remain essential. However, psychological support and marital or fertility counseling are important and the cystic fibrosis center may continue to be a good place for subjects to obtain this help. CASE 2. CILIARY DEFECT.-A 27-year-old married man was referred because of bronchiectasis and recurrent pulmonary infections. His early childhood development was normal, but he had had numerous episodes of croup, ear infections, and possibly bronchiolitis. His health improved in childhood and was considered normal until age 11-12 years, when a persistent but nonproductive cough developed. A chest radiograph was normal. For the next decade of his life he did well and learned to cope with his cough. He continued to have occasional respiratory infections, with several attacks of sinusitis spaced along. In his mid-20s his cough became consistently productive of purulent phlegm and he noted some dyspnea on exertion. A thorough medical evaluation disclosed mild pulmonary obstructive disease, bilateral upper lobe bronchiectasis on a bronchogram, normal serum al-antitrypsin and quantitative immunoglobulin levels, and a normal sweat test analysis. Residual maxillary sinus opacification was evident on radiography. Thereafter, more attention was paid to his respiratory infections. Sputum cultures generally showed mixed flora or at times a predominance of such bacteria as S. pneumoniae, Hemophilus sp., and S. aureus. Pseudomonas was not recovered. Intermittent use of antibiotics seemed to control the lung infections. When the patient was referred several years later for reevaluation of lung status, it was evident that cough and periodic respiratory infections were no longer just a nuisance, but were now 49

causing considerable concern because the process was not being controlled. His health was dragging him down and contributing to the tension and pressure of a competitive sales position. He had been married for about 4 years, and was a bit concerned that a pregnancy had not occurred, although no fertility evaluation had been initiated by the couple.

COMMENT.-Frankly, I thought the patient had a mild form of cystic fibrosis involving primarily the respiratory tract plus the good fortune of not yet having Pseudomonas in his airways, As was true of other patients, the sweat test findings were probably in error and his possible infertility also fit the cystic fibrosis male patient profile. He did not have siblings, so the family history was not helpful. However, as the workup progressed, several findings underscored the possibility that this man’s illness was different from cystic fibrosis. Sweat electrolyte values, reliably obtained and analyzed, were normal, with the chloride value being 30 mEq/L. Sputum cultures for P. aeruginosa were repeatedly negative and serum antibody titers against a panel of Pseudomonas lipopolysaccharide antigens were absent or low, finally convincing us that he had not had much exposure or many infections with this bacterial species. A formal fertility assessment was made. A fresh sperm sample contained only a sli htly reduced total count (15 x 106/ml; E normal, > 20 x 10 /ml), but the sperm were either immotile or poorly so; those that had movement showed uncoordinated twitching and little forward motion. Adult men with cystic fibrosis are often infertile, but their infertility is attributed to oligospermia. Finally, this patient’s lung function and respiratory status remained stable during several years of observation, and the lung disease did not seem to be progressive, as is usually the fate of cystic fibrosis patients, especially once Pseudomonas infections occur. Some evidence indicates that subjects with ciliary defect disease have minimal fall-off in lung spirometric function. Corkey and colleagues163 performed serial pulmonary function tests in a small group of teenage or young adult patients with immotile cilia syndrome during a 4- to 14-year interval. Some air trapping was evident, but FVC!, FEVi,,,, and midflow expiratory rates were surprisingly stable, although each value was somewhat abnormal to begin with. 50

Arterial blood gas oxygen tensions ranged from 60 to 86 torr. Another diagnostic possibility was Young’s syndrome=7, 238 in which males, undergoing infertility evaluation, have azoospermia due to obstruction of the epididymis by inspissated secretions and chronic sinopulmonary infections usually of a mild and self-limited degree. This syndrome does not feature any ciliary ultrastructural defects or a salt losing abnormality that distinguish the dyskinetic ciliary diseases and cystic fibrosis. Eventually, we investigated the patient’s ciliary clearance and the ultrastructure of his nasal cilia. A specimen of cilia was obtained with a mucosal scraping. The method, as described by Reynolds and Malech,164 is quick and simple and not traumatic for the patient. Spray a small amount of tetracaine hydrochloride (Pontocaine) into one nostril. After a few minutes, spread the nares with a nasal speculum: scrape the inferior turbinate with a metal comedone extractor-a metal rod with a 3-mm-diameter spoon at the end. The edges of the spoon are thin but not razor sharp, since the object is to scrape off surface epithelium without cutting the delicate tissue. When the inferior turbinate is well visualized, insert the end of the instrument to at least the central bulge of the inferior turbinate. Pressing gently, but so that some resistance is felt, draw the instrument forward, scraping about 1 cm of the inferior turbinate’s surface. Withdraw the instrument and place the end containing the tissue sample into a solution of O.lM sodium cacodylate (arsenate) buffer containing 2% glutaraldehyde. The solution should be at room temperature. This buffer and fixative, usually obtained from whomever does the electron microscopy, is stored at 4” C and warmed to room temperature just before use. Allow the small piece of tissue adherent to the inner edge of the instrument to fix for about 1 minute. Then use a stiff wire to tease the sample off the spoon and into the liquid. Let the sample fix for another 30 minutes after which it may be stored in O.lM sodium cacodylate buffer for as long as necessary before embedding. After the sample is dehydrated and embedded in any of the available resins used for electron microscopy, thick sec51

tions are cut to locate ciliated cells. Although scraping, unlike biopsy, results in a loss of normal tissue orientation, there is actually an advantage to that. In order to interpret their ultrastructure, the cilia must be cut in thin sections across the axoneme as close to perpendicular as possible. Even slightly oblique cuts result in blurring of detail. In the scraped specimens there are always some correctly oriented cilia. The biggest problem may be locating a cytopathologist skilled in interpreting the fine parts of ciliarl anatomy; some of the pitfalls are discussed elsewhere.@’ 7 In our patient, the predominant lesion was an absence of radial spokes. This lesion is one that can occur without affecting the rotation of viscera, so situs inversus is not usually present. There was no clue from the location of the cardiac silhouette on the chest radiograph that this condition might exist. CASE 3. ACQUIRED HYPOGAMMAGLOBULINEMIA.-A 66-year-old retired executive had known that he had chronic bronchitis and mild obstructive lung disease for the past 10 years, which was attributed to prior cigarette smoking; he was not incapacitated. Six years earlier some “streaking” on a chest radiograph had been evaluated with rigid bronchoscopy and he was told that it revealed lower lobe bronchiectasis. For the past 4 years he had had one episode of pneumonia each winter that did not require hospitalization and was treated with oral antibiotics. The patient produced about 1 cup of watery sputum per day, did daily postural drainage and deep breathing exercises, and intermittently took ampicillin or tetracycline if his sputum became more purulent. Except for mild hypertension that was controlled with a thiazide diuretic, he had no other important health problems. In July, a cold developed and settled in his chest and persisted, causing cough, malaise, and fever, despite oral ampicillin therapy. He was hospitalized and was found to have a left upper and a left lower lobe pneumonia, high fever (104”-105” F), shortness of breath, and&ft pleuritic chest pain. Sputum cultures were not diagnostic. He was placed-o-h-dose intravenous penicillin G, 5 million units every 6 hours, for several days; since little improvement occurred, chloramphenicol was instituted, and during an B-day period his fever gradually decreased. Two days after the antibiotic was withdrawn, however, his temperature abruptly spiked and his pneumonitis recurred. He was transferred to another hospital. A chest radiograph showed an extensive pneumonic process in52

volving the left upper and lower lung and a right middle lobe infiltrate, too; his white blood cell count was 40,000 cu mm, with a marked shift to the left. Analysis of a transtracheal aspirate showed many polymorphonuclear granulocytes and predominantly gram-positive cocci; that culture and several blood cultures yielded S. pneumoniae type 7. Initially penicillin G and gentamitin were begun; when the culture results became available, only penicillin was continued, 2.4 million units/day. He slowly improved and defervesced during a lbday period. Within 2 days after antibiotic therapy was stopped the patient’s temperature again spiked and his illness recurred, exactly as before, including the positive pneumococcal cultures. More immunologic investigations were done. Serum protein electrophoresis showed zero percent y-globulins and low serum immunoglobulins (IgG, < 2 mgidl; IgM, < 4 mg/dl; IgA, < 5 mg/dl). A diagnosis of primary agammaglobulinemia was made and he received multiple units of fresh frozen plasma to elevate his serum IgG level to about 200 mgidl. He received parenteral penicillin for another 14 days and gradually improved. Again, however, within 24 hours after antibiotic therapy was stopped, his temperature spiked to 104” F rectally and sputum cultures were positive for S. pneumoniae. Since the bacterium was quite sensitive to penicillin, this antibiotic was used again, plus frequent plasma transfusions to keep the serum IgG level above 250 mgidl. This time the regimen was successful: the pneumonia was controlled and no relapse occurred. Later the patient received a more detailed immunologic workup that disclosed a normal peripheral WBC count and differential, a normal percentage of T lymphocytes (80%), but a slightly low percentage of B lymphocytes (6%). T suppressor cell studies were not done. Delayed skin hypersensitivity tests were normal.

COMMENT.-Although this patient had chronic and recurrent pulmonary infections, including a yearly episode of pneumonia for the past 4 years, they evoked no particular concern in the physician for there was an established underlying diagnosis of bronchitis and bronchiectasis, antibiotics readily controlled the bacterial infections, and the patient was reasonably well and not debilitated. The last episode was clearly different in that several prolonged and repeated courses of specific antibiotic therapy could not eradicate the encapsulated gram-positive coccal organisms. Realizing that the situation was unusual, the physician undertook a broader investigation of the immune system, which revealed a deficiency in the three major serum im53

munoglobulin classes. Not until immunoglobulin replacement was added to the therapeutic regimen was control over the pneumococcal pneumonia and sepsis achieved. Presumably the patient had common variable hypogammaglobulinemia, probably an acquired form, although his prior immunoglobulin status was not known with certainty. Subsequently he required periodic injections of yglobulins or plasma transfusions at 4 to 6 week intervals to maintain a minimal IgG level of about 300 mg/dl, and he did not have another major infection during the 4 years he received this therapy. He continued to require a short course of oral antibiotics if his bronchitis flared up, and he practiced daily postural drainage to facilitate removal of secretions. Among the antibiotics tried, the trimethoprimsulfamethoxazole combination seemed particularly helpful in this patient, and for several years he took one tablet twice (80/400 mg) daily. Whereas the cellular immune details of hypogammF&obulinemia are fascinating and are being worked out, for practical purposes therapy for the disorder is limited to periodic, passive replacement of immunoglobulins. Since large amounts of y-globulin must be given to raise serum IgG levels to 200-500 mg/dl, the minimal range associated with control of pyogenic infections, the intramuscular route and prospects of monthly injections are not popular with most patients. Plasma infusions are preferred. Even though plasma provides thorough immunoglobulin replacement since all five classes can be delivered, the increment and persistence of levels of IgM and IgA may be small and transient compared with levels of IgG. One drawback to plasma therapy is acknowledged. There is a risk of transmitting hepatitis B virus or acquired immunodeficiency syndrome. The risk can be minimized greatly by using known or screened donors who are free from the infection. The irony of this patient’s course was an infectious complication in another organ. He was managed very successfully with periodic plasma transfusions, but because of the possible risk of an opportunistic infection transmitted with plasma, he was switched to an intravenous y-globulin preparation. Perhaps this alternative therapy was begun too late, because within a few months of the last plasma trans54

fusion, a low-grade smoldering hepatitis became evident. Eventually, hepatitis and liver failure caused the patient’s death. In summary, many chest patients have recurrent sinopulmonary disease which is engrafted on underlying chronic bronchitis, intrinsic asthma, or bronchiectasis. Usually infections behave in a predictable way and acute exacerbations with occasional episodes of frank pneumonia are expected. With intensive pulmonary toilet, bronchodilators, and simple antibiotic regimens, the patient usually returns to a recognized baseline condition with some cough, expectoration, and wheezing. Little immunologic investigation is warranted. Perhaps quantitative serum immunoglobulin analysis or a protein electrophoresis is indicated at least once for a record when routine blood work is being done. IgG subclass values are of interest, if these can be measured easily, because of very specific deficiencies of IgGs and IgG4, sometimes coupled with IgA deficiency. Yearly use of currently recommended influenza1 vaccines is advisable, and immunization with a polyvalent pneumococcal vaccine at 3-year intervals is justified.‘@ If the frequency of infection increases or an infection of unusual severity occurs, as illustrated in this case, further investigation of host immunity should be considered. Replacement therapy can be very specific and effective. HOST DEFENSES OF THE ALVEOLAR

SPACES

To recapitulate (see Table 1 and Fig 1) what has been said about the defenses in the naso-oropharynx and along the conducting airways, mechanical barriers, the cough reflex, branching of the pulmonary tree to aerodynamically filter-inspired air, and mucosal mechanisms (ciliary clearance, local immunoglobulins, and mucus secretions) all are coordinated to eliminate foreign substances entering the respiratory tract. These defenses are not perfect in that they may not quantitatively eliminate all particles inspired into the lungs, but their overall effectiveness is excellent. Airways distal to the major bronchi are probably sterile in normals. However, some particles of small size and certain geometry can elude all of the above-named 55

mechanisms and reach the air exchange surface of the alveoli spaces. When this occurs, another group of host defense factors must take over. Anatomically, lung structure changes at the level of respiratory bronchioles, and in the terminal units (alveolar ducts and alveoli) ciliated epithelium and mucus-secreting cells (goblet cells and mucus glands) are no longer present. Therefore mucociliary clearance does not exist, nor does coughing effectively clear material from the alveoli. Microbial clearance and the removal of other antigenic material from alveoli depend entirely on cellular and humoral factors. These include phospholipid surfactant and proteins (immunoglobulins and complement factors) in the alveolar lining material and phagocytic cells, i.e., alveolar macrophages and PMNs. Inhaled or aspirated bacteria are an appropriate example. If a bacterium of critical size (0.5-3 p. in diameter) is deposited in an alveolus, the microbe may encounter at least three substances that conceivably could inactivate it, exclusive of its eventual inactivation by phagocytosis. The actual pathway the aerosolized microbe takes is not certain, but it may have to contact the alveolar wall, roll and tumble along in the alveolar lining fluid (or cling and remain stationary?), picking up a coat of the various soluble lipoprotein substances present, encounter a patch of IgG or a complement factor (C3b) or possibly some nonimmune opsonin like a fibronectin fragment, dodge around a proteolytic enzyme that is contending with an antiprotease, and finally keep squirming away from some phagocyte trying to catch it. Nevertheless, the chase does not last very long, and an alveolar macrophage captures the bacterium within a few minutes. With experimental murine models in which bacteria are aerosolized into the lungs and lung histology is then examined soon after, bacteria do not remain free on the alveolar surface but are almost quantitatively ingested by macrophages.167-16g Mice, when exposed to an aerosol cloud of bacteria (S. aureus or Proteus mirabilis), will respire low numbers of organisms into their alveoli (about 10% of a 2 x lo7 organism-dose per cubic foot with 30 minutes of exposure, but up to 36% of a lower inoculum of lo5 organisms).167 Within a few minutes, staphylococci are al56

most all within alveolar phagocytic cells; the ing:stion of Gramgram-negative organisms, is somewhat slower. negative bacteria are usually cleared from lungs more slowly.16g Of particular importance is the respective activity and specificity of alveolar space opsonins. First, surfactant, secreted by type II pneumocytes, may have antibacterial activity against staphylococci and rough colony strains of some gram-negative rod bacteria. Second, immunoglobulins, principally of the IgG class and, in lesser concentration, monomeric and secretory forms of IgA, may have specific opsonic antibody activity for the bacterium. Third, complement components, especially properdin factor B, might interact with the bacterium and trigger the alternative complement pathway. One or all of these possibilities can prepare the bacterium for ingestion by an alveolar macrophage, or the active complement sequence can lyse it directly. Although alveolar macrophages avidly phagocytose some inert particles, they may ingest viable bacteria with considerably less enthusiasm. Coating or opsonizing the organisms will enhance phagocytosis approximately tenfold in an in vitro culture system. IgG is capable of selectively enhancing alveolar macrophage phagocytosis, and complement can function in concert with IgG to enhance or amplify the process. The effect of nonimmune opsonins must be considered as well. Certainly surfactant, as mentioned, has a role, as well as fibronectin and cytophilic IgG. Phagocytosis, defined as the ingestion of particulate matter by cells, is a complex process that is divided into two phases: attachment of the particle to the cell surface and internalization.17’ Attachment of the particle to the surface of the phagocytic cell appears to be essential prior to ingestion. This binding may occur at random but is greatly enhanced by opsonization of the particle by antibody (especially IgG) or one component of the complement system, C3b. Opsonin-dependent phagocytosis is mediated by receptors on the cell surface for immunoglobulin or complement. Receptors for the Fc portion of IgG and for the third component of complement (C3) have been identified on human monocytes171- 73 and alveolar macrophages.‘14 There is 57

considerable evidence that the number and function of these receptors can be modulated by lymphocyte products and other mediator substances. Ingestion of membranebound particles occurs via a process that is energy dependent and involves the actin-myosin system of the phagocytic ce11.17’ The plasma membrane of the ingesting cell surrounds the bound particle, enclosing it in an endocytic vesicle. Phagocytic cells have well-developed mechanisms that operate to kill internalized pathogens. Fusion of the vacuole (or phagosome) containing an engulfed organism with lysosomes within the cell follows the actual ingestion of the pathogen. This process exposes the pathogen to hydrolytic enzymes and other bactericidal proteins, as well as myeloperoxidase, an enzyme that in combination with oxygen metabolites is extremely active in killing phagocytosed organisms. As will be discussed, several organisms are known to interfere with phagosome-lysosome fusion and are able to promote their own intracellular survival. An important factor of the antimicrobial systems in phagocytic cells is their ability to generate reduced oxygen species, such as hydrogen peroxide, superoxide anion, and hydroxyl radicals. r 4, l7 In response to a phagocytic stimulus, neutrophils, monocytes, and macrophages undergo a respiratory burst that results in increased oxygen consumption, accelerated utilization of glucose, and activation of the hexose monophosphate shunt, with generation of reduced pyridine nucleotide (NADPH) and the production of reduced oxygen metabolites. These have sufficient antimicrobial activity, both independently and in the presence of myeloperoxidase.rT4 Myeloperoxidase (MPO) is a lysosomal enzyme present in abundance in neutrophils and to a lesser extent in monocytes. Macrophages contain little or no demonstrable MPC)+aalthough they may have some other peroxidase activity. MPO reacts with hydrogen peroxide and a halide (usually chloride) to form highly reactive products (especially hypochlorous acid) with potent antimicrobial activity, probably mediated via halogenation and/or oxidation of the surface of the ingested pathogen.175 Even though macrophages lack myeloperoxidase, it is likely that 58

toxic oxygen species remain an important microbicidal mechanism for these cells. Following containment of bacteria, the fate of alveolar macrophages is not certain. They are long-lived tissue cells that can survive at least for several months and presumably are capable of handling repeated bacterial and other microbial challenges (reusable phagocytes). Because they are mobile cells, they can migrate quickly to other alveoli through the “pores of Kohn,” or move to more proximal areas of the respiratory tract (to the region of respiratory bronchioles) and get aboard the mucociliary escalator for elimination from the lungs. In addition, macrophages gain entry into lung lymphatics at this same place and can be carried to regional lymph nodes. This exit gives them access to systemic lymphoid tissue and is important in initiating cellular immune responses. Undoubtedly, macrophages are instrumental in degrading antigenic material and presenting it to appropriate T lymphocytes in these nodes. Increasingly, attention is being given to the effector immune role of macrophages.177 The alveolar macrophage has acquired an interesting dual role in the respiratory tract-that of an affector cell, to phagocytose debris, to process foreign antigens, and to kill ingested microorganisms, and that of an effector cell, to initiate immune responses and the inflammatory reaction. We will discuss later the identification of chemotactic factors secreted by alveolar macrophages that may be important in generating parenchymal inflammation and attracting PMNs to the lung. Alveolar macrophages can usually inactivate microorganisms and host defense surveillance is successful; clinical disease and pneumonitis rarely develop. However, if a sufficiently large bacterial inoculum reaches the lower respiratory tract, or if particularly virulent microorganisms are inhaled, the macrophage system can be overwhelmed. PMNs are recruited to help out. This situation points out the dual phagocytic protection present in the alveolar spaces and raises the intriguing question of how the influx of other phagocytic cells is initiated and modulated. 59

INTERPLAYBETWEENALVEOLARMACROPHAGESAND POLYMORPHONUCLEARGRANULOCYTES Alveolar macrophages are the most numerous resident phagocytes present in the alveoli (about one cell per three to four alveoli). They are the bona fide first line of cellular defense on the airside of the lower respiratory tract. A few PMNs, about one per 100 alveoli, are present, but primarily they are reserve phagocytic cells close by in the intravascular compartment. A plentiful supply of PMNs resides in the blood of lung capillaries as part of the body’s pool of marginated PMNs. Even though PMNs are in close proximity to alveolar spaces, they are nonetheless separated by several planes of tissue: capillary endothelium, interstitial space and alveolar epithelium. Depending on the species of bacteria that are inhaled into the lungs, alveolar macrophages and/or PMNs are selected to respond to the inoculum. Experimentally in mice, a small dose of aerosolized S. aureus is contained solely by macrophages, whereas Klebsiella and Pseudomonas evoke a PMN exudate in alveoli.178 Green and Kass168 noticed also that staphylococci were contained by macrophages and there was no histologic evidence of an inflammatory response. To test the concept that two sets of phagocytic cells in the lungs, alveolar macrophages and polymorphonuclear granulocytes, are required to defend the lungs against inhaled bacteria, an aerosol inhalation mouse model (Bale/c strain) was used.17’ Mice were rendered selectively granulocytopenic with a heterologous antiserum, raised in rabbits against peritoneal exudate granulocytes, to determine the relative contribution of these two cells types in intrapulmonary bacterial killing of three organisms: S. aureus, K. pneumoniae, and P. aeruginosa. The clearance of staphylococci was unimpaired in granulocytopenic animals, confirming the primary role of the alveolar macrophages in the killing of these organisms. In contrast, granulocytopenic animals cleared only 10% of an inoculum of Klebsiells, compared with 33% clearance in normal animals, and Pseudomonas proliferated to 513% of baseline levels in graulocytopenic animals, whereas normal mice cleared 27% of the inoculum. These findings indicated that circu-

60

lating granulocytes play a major role in the clearance of the latter two organisms. Gram-negative bacteria seemed to require more of a PMN phagocytic cell response for containment or clearance from the lungs than gram-negative species. Perhaps this is related to cell wall products, such as lipopolysaccharide endotoxin, that these gram-positive organisms contain, or to release of unidentified exotoxins. Certainly, patients and animal models that are immunosuppressed to the point that circulating blood PMN levels are low or absent are at significantly increased risk of infection, especially bacterial infection with aerobic gramnegative rods and with certain fungi such as Aspergillus sp. Because normal oropharyngeal flora can be aspirated into the lungs and cause pneumonia, the pulmonary clearance of representative species of normal pharyngeal flora (Streptococcus sanguis, Streptococcus salivarius and N. catarrhalis) was determined after aerosol inoculation of bacteria into the lungs of normal Balb/c mice.17’ Viable bacteria were quantified in lung cultures 1, 2, and 4 hours after challenge and the phagocytic cell response was characterized by enumerating the cells retrieved by bronchoalveolar lavage. For the two species of streptococci, about 4 x lo5 bacteria were initially deposited in the lungs of each mouse, and 5% or less of the dose was found viable in lungs at 4 hours. Whereas S. sanguis was associated with a twofold increase in alveolar macrophages in lavage fluid but no increase in PMNs, S. salivarius caused a similar increase in alveolar macrophages and a 20-fold increase in PMNs such that about one half of the respiratory cells recovered by lavage were PMNs. In contrast, N. catarrhalis did not affect the number of alveolar macrophages but elicited a 400-fold increase in PMNs. Thus, normal pharyngeal organisms are cleared rapidly from the lung, but by a dual phagocytic cell mechanism. In these murine models, the aerosol route for delivery of bacteria into the lung will deposit only a small inoculum of about lo5 organisms. With larger numbers of bacteria, administered by a bolus method into a selective portion of the lung,lsO the pattern of phagocytic clearance can be 61

changed, however. For staphylococci, when a small inoculum is given (lo5 bacteria into approximately 20% of the lung volume of a mouse), alveolar macrophages still ingest and clear these bacteria promptly, as described.168X 17’ With progressive increments of a staphylococcal inoculum (10610’ organisms), more PMNs are recruited into the alveoli to cope with the bacteria, and inflammation is more extensive. Thus, many factors may govern the kinetics of the macrophage-PMN interchange in the lung. Certainly the species of organism, virulence, and inoculum size are all important for lung clearance in the normal, nonimmune animal. Of course, we have been describing a murine model and therefore the findings might not apply strictly to the human, but this extrapolation seems plausible. The lung is capable of mounting an extensive inflammatory response, which is a potent mechanism to augment host defenses (see Table 1). The development of inflammatory response and hence pneumonia is a deliberate and controlled reaction in the lungs. The ingredients of initiation, amplification, and, finally, suppression are present.“’ When the lung parenchyma mounts an extensive inflammatory response it may be perceived as clinical illness, and a chest roentgenogram usually reveals an infiltrate. Granulocyte movement into the alveoli is an orderly reaction initiated from the alveolar side. This is termed directed migration or chemotaxis. At least two mechanisms for chemotactic activity exist that can set in motion the inflammatory response in the alveoli and amplify the PMN response. The first is best illustrated with the example of gram-negative rod bacteria known to contain lipopolysaccharide substances termed endotoxins. Some complement components, particularly factor B, are present in small amounts in bronchoalveolar fluids. Bacterial endotoxin can directly activate the alternative complement pathway, leading to the formation of fragments such as C5a that are known to be potent stimulators of PMN chemotaxis. In addition, the inflammatory response may include activation of the kinin system. This could result in generation of kallikrein, which has chemotactic activity, and bradykinin, which is capable of increasing vascular permeability and could account for the accumulation of fluid and other hu62

moral substances in alveoli that accompanies pneumonia. The second mechanism may emanate from the alveolar macrophage itself. Following phagocytosis of opsonized bacteria, chemotactic factors are synthesized and secreted that will selectively attract PMNs. In vitro and in vivo studies with macrophages from monkeys’s’ and guinea pigs1s3* 184 have shown that following phagocytosis of opsonized bacteria, a chemotactic factor is synthesized and secreted which selectively attracts PMNs. Such a factor is of small molecular weight (< 5,000 daltons), heat labile, and unaffected by antisera to complement components C3a and C5a, which neutralize their respective activities. Although this factor was identified first in macrophages of monkey and origin, it has now been found in human cells as g-g%!;6 This mechanism would permit alveolar macrophages to recruit secondary phagocytes, PMNs. Once PMNs and other components of edema fluid have filled alveolar spaces, an exudative inflammatory reaction exists in lung parenchyma, and pathologically pneumonitis is present. Ultimately, lung tissues become consolidated. CHEMOTACTIC FACTORS PRODUCED BYHUMAN ALVEOLAR MACROPHAGES Once it was discovered that monkey and guinea pig macrophages produced chemoattractant substances, the observations were extended to human alveolar macrophages. Merrill and colleaguesls5 found that cell adherence, versus nonattachment, in tissue culture conditions was sufficient to create chemoattractant activity in cell-free supernatant after 22 hours, but if an IgG-containing immune complex or microbial stimulus was given to the macrophages, production of chemotactic activity was accelerated greatly. Like macrophages from other species, the chemoattractant substance(s) was relatively specific for inducing PMN motility and decidedly less potent for mononuclear cells. To characterize the chemotactic activity produced by monolayers of alveolar macrophages from non-cigarette smokers, supernatant specimens from unstimulated cell cultures were collected, concentrated, and chromatographed on a calibrated Sephadex column (Fig 4). Eluent 63

Chymotrypsln V, MW 25,000 ’

i

Ribonucleose MW 13,700

Fig 4.-Gel filtration of pooled alveolar macrophage culture supernates. One hundred fifty ml of unstimulated AM culture supernate from nonsmokers were concentrated to 7 ml and filtered through Sephadex G50 SF (column dimensions 2.6 (i.d.) x 61.4 cm with gel bed volume of 324.6 ml) in phosphate-buffered saline, pH 7.2. The position of eluent fractions (4.5-ml fractions were collected) is expressed as VE/VT. A, relative protein content (A& of the fractions and the elution position of calibration markers. B, migration of PMNs in micrometers toward these fractions. Control cell migration in the buffer was 30 f.~. The column apparatus was sterile and eluent fractions were free of detectable endotoxin material. (From Merrill et aLis Reproduced by permission.)

fractions containing chemotactic activity for PMNs centered about the elution position of a 9,500-dalton material and an approximately l,OOO-dalton marker. In similar studies using supernatant from zymosan-stimulated macrophages, the same two peaks of chemotactic activity were 64

detected in the elution profile, but the relative potency of the activity was somewhat different, shifting to more of the smaller molecular factor. This suggested that acute cell activation might change the relative proportions of factor secretion or indicate that the smaller substance was a breakdown product of the larger. The relationship between these two possible chemotactic factors was not elucidated completely. Further analysis of the larger molecular weight factor indicated it was susceptible to proteolytic degradation with trypsin that diminished its activity. Isoelectric focusing in polyacrylamide gels of an lz51 trace-labeled specimen actually revealed the “homogenous” factor to disperse into a multiple band profile, but with chemoattractant activity confined to a single peak with an isoelectric focusing point of 5.0. Various inhibitors, including anti-C5 and anti-C3 antisera, confirmed the integrity of this 9,500-dalton chemotactic factor to be noncomplement in nature. But blocking prostaglandin metabolism in the cells did inhibit early release of chemotactic activity by macrophages, and it was considered probable that some activity attributed to the small molecular weight substance could represent a lipoxygenase pathwa product. Concomitantly, Hunninghake and colleagues18 J were analyzing their human alveolar macrophage-derived chemotactic factor. The results were complementary but slightly different, although the basic methodology used was generally similar. Bronchoalveolar lavage of healthy nonsmokers recovered respiratory cells, principally alveolar macrophages (mean, 92%), that were cultured and stimulated with a variety of particles, opsonized particulates, and immune complexes (bovine serum albumin-IgG antiBSA antibody). Active cell supernatants were chromatographed through Sephadex G25 or Biogel P2 gel media in phosphate-buffered saline. Cell supernatants were generally harvested within 3 hours after specific stimulation for assay. Specific macrophage stimulation could enhance the release or secretion of chemotactic activity in several hours; however, with extended culture (18 hours), nonstimulated control cells eventually produced maximal activity. All particles stimulated chemotactic activity versus controls, but opsonization of the particles provided an addi65

tional increment, especially with heat-killed S. aureus. IgC-containing complexes were particularly potent, but complement receptor attachment to the macrophages’ surface was not a potent stimulant. After fractionation of active cell culture supernatants, chemotactic activity was consistently found in the 400- to 600-dalton elution range of the calibrated columns. Detailed characterization of this material showed it to be stable on heating at 56°C and lOO”C, to exhibit stability over an enormous pH range (pH 1.0-12.01, to resist a variety of proteolytic enzymes, to contain two major isolectric points (PI 7.6 and 5.2) and to be extracted by organic solvents, suggesting it contained some lipid components. In terms of functional activity, the material was preferentially active for directing migration of PMNs compared with that of monocytes and eosinophils. Moreover, macrophage supernatants containing chemotactic activity induced normal human PMNs to release lysozyme and lactoferrin. In summary, several small molecular size chemotactic factors have been conclusively demonstrated and characterized. It seems that human alveolar macrophages can secrete at least two well-defined chemotactic factors that show selective activity for PMNs. The small, less than l,OOO-dalton factor is in part a lipid-containing substance and may represent a lipoxygenase pathway metabolite of arachidonic acid. This possibility is relevant because one such substance, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid, has been found to stimulate random and directed migration of PMNs and eosinophils. At least two groupsls7, ls8 have reported that human alveolar macrophages produce LTB4, which is a chemotactic substance. DEFECTS IN ALVEOLAR MACROPHAGES THAT CONTRIBUTE TO PNEUMONIA To our knowledge, genetic or inherent defects in alveolar macrophages have not been described that selectively affect the lung and are not part of a more general problem with the lymphoreticular system in which blood monocytes and other tissue macrophages are involved. As tissue mac66

rophages are found in liver (Kupffer cells), skin (Langerbans cells), spleen, bone (osteoclasts), peritoneal cavity, and the CNS, lung macrophages represent just one species of these cells. It has been demonstrated that alveolar matrophages from subjects with cystic fibrosis have normal phagocytic and antibacterial function,‘“” for this disease was an example of possible genetically impaired cells. Exogenous factors within the alveolar milieu can have profound effects on macrophage function, however. An example is alveolar proteinosis, an acquired lung disease in which the accumulation of surfactant-like material, probably due to poor regulation of alveolar type 2 cells, builds up in alveolar spaces and impairs gas exchange. This disease features macrophages that engorge themselves trying to eliminate the excess lipoprotein substances. Inadequate clearance of surfactant rather than overproduction is the apparent defect. A striking clinical feature of alveolar proteinosis is a propensity for infection with unusual microorganisms, particularly Nocardia. Studies of alveolar macrophage function in patients with this disorder have helped elucidate the pathogenesis of these infections.lsg, lgo Macrophages obtained by lung lavage from these patients contain large quantities of periodic acid-Schiff positive staining lipid/protein material that is presumably ingested within the alveoli. These cells have been demonstrated to have a variety of functional defects. Adherence to glass and chemotactic function are impaired, and intracellular killing of Candida pseudotropicalis is defective, though ingestion appears unaffected. In another report,lgl the phagocytic capacity for C. albicans was reduced. The functional activity of peripheral blood monocytes is normal in patients with this disorder, suggesting that the derangement in alveolar macrophage function is acquired in situ and may be related to ingestion of the lipid-rich alveolar exudate. If normal alveolar macrophages are forced to ingest the lipoprotein material, they too will develop these functional defects. Ingestion of globules of lipid crowds the cytoplasm of the macrophage so that there is not adequate room for lysosomes and lysozomal enzymes; thus, decreased lysozomal enzyme killing is an acquired defect in an overstuffed macrophage. 67

Although immunosuppression of the host with corticosteroids or cytotoxic drugs will affect lung macrophages, identification of a specific lesion has been difficult. In a natural disease situation of monocytic leukemia in which the precursor source of blood monocytes that supplies potential tissue macrophages was absent, the effect of prolonged monocytopenia in reducing the number of alveolar macrophages was negligible.ig2 The reason the lung supply might not have been depleted was that a low level of macrophage replication was detected, suggesting that a form of in situ cell proliferation might occur to maintain the macrophage population.lg3 The functional activity of these macrophages, once removed from the leukemic host, was normal in vitro. With animal models, the kinetics of macrophage depletion and repopulation can be examined more closely. In dogs, bone marrow was obliterated by total body irradiation and then replaced with autologous marrow infusion from donors of opposite sex.i’* Seven days after irradiation, monocytes had disappeared from the blood, and by 3 weeks, the number of recoverable alveolar macrophages from lung lavage had decreased almost 50% from baseline values. By 30 days, new macrophages had returned to the air spaces (identified by sex chromatin changes and MPO staining that denoted a younger, monocyte type of cell). A similar reduction in lung macrophages was found in another canine model of irradiation or when cyclophosphamide treatment was used.lg5 Poor motility could be a factor. In seven humans with smoke inhalation-related mild lung injury, alveolar macrophages responded sluggishly to a chemotactic stimuluslss; this suggested that impaired movement of these cells might be a factor in the susceptibility to lung infection that patients with smoke exposure and the adult respiratory distress syndrome seem to have. In a guinea pig model using cyclophosphamide or cortisone acetate to cause immunosuppression,ig7 Pennington and Harris noted that alveolar macrophages in culture produced less chemotactic activity (about 25% less than control cells) in their cell culture supernatants. Perhaps immunosuppression can decrease the secretion of chemotactic factor(s) and blunt the PMN inflammatory response. This was found to be the case 68

when a P. aeruginosa inoculum was delivered to the lung in cyclophosphamide-myelosuppressed guinea pigs, for chemotactic activity generated in bronchoalveolar lavage fluids was significantly reduced.l” This chemotactic activity was not associated with a complement fragment but had characteristics similar to the macrophage-derived factors described before. In summary, lung macrophages can be affected by general regimens of immunosuppression in patients so treated, or in those with malignancy that impairs bone marrow function, for opportunistic lung infections are greatly increased in these groups as a whole. However, it is difficult to separate the contributions of granulocytopenia and a potentially diminished inflammatory response and an abnormal pattern of mucosal colonization with pathogenic bacteria from the specific effects that immunosuppression may have on macrophages in arriving at the probable cause for a nosocomial pneumonia. However, it is reasonable to conclude that mild or intermittently administered cytotoxic regimens may not deplete the population of lung macrophages very much, for the longevity and durability of the cells are such that they can survive or replicate in situ when general bone marrow function is poor and a ready supply of precursors is limited. Subtle effects of immunosuppression may be harder to discern or to quantify if these drugs cause macrophages to alter their secretion of mediators or decrease bactericidal activity. More sensitive assays will be required to detect such changes, if any practical clinical tests are developed that might predict which immunosuppressed patients are most vulnerable to infection. Availability of Immune Opsonins As discussed, alveolar macrophages have special cell membrane receptors for IgG, and this class of antibody seems to be the most effective opsonin for enhancing phagocytic uptake of bacteria. Individual IgG subclasses, when fabricated into immune particle complexes, bind to macrophages with varying avidity; IgGs and IgGi do so in greater numbers than the other two subclasses. However, selective absence of serum IgG, or IgGs and IgG,, often in association with IgA and IgE deficiencies, correlates well 69

with recurrent sinopulmonary bacterial infections. Patients with multiple myeloma and an excessive level of serum immunoglobulins are very susceptible to pneumonia, which accounted for about half of the infections in one group of patients reported and was the cause of death in 20% of them.lgg The increased frequency and poor outcome from infection in patients with myeloma may stem from two causes: a reduction in immunologically competent globulins, which decreases serum opsonic efficiency, and an impaired antibody response to bacterial antigens. This functional loss of opsonic activity can be contrasted with the case presented earlier of the man with acquired hypogammaglobulinemia and pneumococcal pneumonia and sepsis who effectively lost his antibody opsonins. Brunhamella catarrhalis, formerly known as Neisseria catarrhalis, may not be just a benign part of the normal nasal flora but a cause of pneumonia. This infection has been found in patients with immunoglobulin abnormalities, such as multiple myeloma,200 and in elderly patients, who may be susceptible because of a diminished immunoglobulin level in serum because of advanced age. As very little has been published about aging and the decline of normal humoral immunity,“l or bacterial clearance from the lung,202 the impact of a functional decline in opsonic antibody and susceptibility to specific infections is an unexplored area. Certainly, the efficacy of immunizing certain patients with pneumococcal polysaccharide antigens is s;;d, and these immune opsonins do help prevent infection. Therefore, it seems desirable to have opsonic antibodies available in serum and perhaps on mucosal surfaces for added protection against encapsulated bacteria and to bolster phagocytic host defense. Role of Nonimmune Opsonins Contained in the alveolar lining fluid are several substances that can mix with inhaled microorganisms or particles as they slide or bump along the alveolar walls. Surfactant204’ 204a and fibronectin are the ones best characterized; both have been shown to act as opsonins. Surfactant also has antipneumococcal activity, for phospholipid extracts and lamellar bodies purified from lung lavage fluid 70

of rats could kill and lyse these bacteria.204b A lysophospholipid, palmitoyl lysophosphatidylcholine, which represents a minor component of the lipids in surfactant, seemed to be the most active fraction. Activity was directed toward gram-positive bacteria, and gram-negative rods resisted the bactericidal effect. It is not certain that actual deficiencies develop in the host that would be sufficient to promote lung infection, but the possibility exists. Surfactant, for example, is diminished when acute lung injury occurs or hyaline membrane disease is present, and preliminary evidence indicates that the composition of surfactant is abnormal in patients with a farmer’s lung type of hypersensitivity lung disease.205 Fibronectin can be detected in normal lung lavage secretions and seems to be a bona fide component of the milieu of the alveolar surface. The origin of fibronectin is probably multifocal since it is in plasma (400,000 daltons sized molecule) but might not diffuse readily across the blood-air alveolar barrier. It is a product of alveolar macrophage secretion; hence it can be added directly to the lung secreconcentration is increased in the lations.206 Fibronectin vage of normal smokers207 and fibronectin values are increased in a variety of patients with forms of interstitial lung disease in whom altered permeability of the capillaryalveolar interface does exist and inflammation (alveolitis) is present.208 The function of fibronectin in the lung is uncertain. This and other related glycoproteins (laminin) are adhesive surface proteins found in plasma and in extracellular matrices. Fibronectin probably helps young, differentiating, multiplying cells stick together in tissue or adhere to a structural support like collagen. Fibronectin is secreted by fibroblasts and, as mentioned, by alveolar macrophages. As fibronectin is an adhesive substance, it is not unexpected that particles or microorganisms become coated and emmeshed in it (opsonin) and attachment to phagocytic cells is facilitated. Czop and colleagues2” studied human alveolar macrophages to find out if they had an opsonin-independent phagocytic mechanism that was specific for particles that activate the alternative complement pathway; such a cell receptor unit exists on human PMNs and blood mono71

cytes and is present on mouse peritoneal macrophages. This mechanism of phagocytosis could be important for lung macrophages which may reside in an alveolar milieu relatively deficient in immunoglobulin opsonins. Alveolar macrophages from healthy cigarette smokers were used and compared with blood monocytes. The particle activator of the alternative complement pathways was zymosan. Monolayers of cultured macrophages avidly ingested zymosan particles, but with trypsin treatment of the cells, this ingestion diminished by more than 50%. In contrast, macrophage ingestion of immune IgG-coated particles was not diminished by the trypsin treatment, reflecting the still intact function of the Fc y receptor. Purified plasma fibronectin, used as an intact 440,000-dalton molecule, would not augment zymosan ingestion, whereas two fragments of 220,000 and 180,000 daltons did enhance, in a dose-dependent fashion, the ingestion of the particulate activator substances. These fibronectin fragments had no augmenting effect on immune Fc receptor-mediated ingestion. Summarizing the results, a trypsin-sensitive membrane recognition unit mediates phagocytosis of particulate activators of the human alternative complement pathway and is present on human alveolar macrophages. Fragmented human plasma fibronectin augmented this capacity, suggesting a nonimmune mechanism might exist for the clearance of some microorganisms from the opsonin-deficient microenvironment of the lung. Noteworthy is the fact that intact (400,000 daltons) native plasma fibronectin was not effective in promoting alveolar macrophage phagocytosis of particulate (rabbit erythrocytes and zymosan used) activators of the alternative complement pathway or of IgGcoated particles. Only fibronectin fragments seemed to be the active ingredients. Therefore, with inflammation and liberal proteolytic enzyme activity in the lung, protein degradation occurs and fibronectin fragments are generated. This means assigning fibronectin “opsonins” a secondary role for now in local lung host defense mechanisms. These probably become operational after the initial inflammatory reaction has occurred and protease enzymes are present to fragment fibronectin molecules. Thus, fibronectin fragments may enhance nonimmune opsonin phagocytosis by 72

macrophages (and possibly PMNs) in the inflamed lung. The origin of them could be from secretion by macrophages and fibroblasts and/or by leakage from the plasma into alveoli. The fragments are thought to work by interacting with the trypsin-sensitive membrane receptor on the macrophage which is the putative complement (C3) receptor. Complement-Mediated Clearance Complement is a triple threat for some microbes that enter the lower respiratory tract, but the overall contribution of this system to lung defense is incompletely understood. As mentioned, the alternative pathway of complement can be activated directly by bacteria or particles that are carried into the airways, and this could produce certain phlogistic factors (C3 or C5a) that promote the inflammatory response, or the whole sequence of the complement pathway could be completed, creating lytic activity that might puncture and perforate the organism and kill it outright. With phagocytosis perhaps assisted by C3b, which acts as a receptor-mediated opsonin in the presence of alveolar macrophages, the macrophages can secrete locally complement components that might augment the inflammatory reaction and perhaps enhance extracellular killing of organisms in the alveolar exudate. In a guinea pig model of acute and chronic P. aeruginosa lung infection, alveolar macrophages were found to increase their synthesis of two complement components, C2 and C4, in both phases of infection,210 thus, providing a local source of certain complement factors in the inflammatory exudate. The availability of a systemic source of complement is needed to contain and clear some bacteria from the alveoli, and this complement probably crosses the blood-air barrier after injury and inflammation have occurred. For example, in inbred mice with low levels of bacterial-specific serum antibodies, complement depletion (alternative pathway) was achieved by giving cobra venom factor.211 Four hours after aerosol deposition of S. pneumoniae, complement-depleted animals had cleared only 75% of the initial number of organisms, whereas saline-treated controls had cleared 91%. Aerosolization with P. aeruginosa was followed at the end of 4 hours by a twofold greater growth of organisms in 73

the complement-depleted animals (446% of the original inoculum deposited) than in the saline-treated controls (211% of original inoculum). Clearance of K. pneumoniae and S. aureus was similar in complement-depleted animals and saline-treated controls. Thus, from this model, one is left to conclude that an intact alternative pathway helps a bit in the clearance of pneumococci, but not in the clearance of S. aureus or K. pneumoniae. This mouse lung model did not seem suitable for P. aeruginosa, since the control mice were unable to handle the aerosol inoculum, and Pseudomonas colony counts increased more than twofold during the 4 hours of observation. In terms of a specific complement component, C5a may be required for generating a maximal inflammatory reaction in the lungs. Its requirement is bacterial species dependent. Still using a murine model, P. aeruginosa were instilled into the peripheral lung of mice with sufficient C5 or congenitally deficient in C5.212 In the early hours (6 hours) after challenge, the mice with C5 mounted a better inflammatory response, contained the number of bacteria, and survived better, which suggested that in the initial phase of Pseudomonas inoculation, C5 was an important factor to have. In a similar model of C5-deficient and C5sufficient mice, S. aureus was inoculated into the lung.213 With this bacterial species, the complement-deficient mice did just as well as the others by generating a comparable inflammatory response, producing a comparable amount of airway chemotactic activity in lung lavage fluids, and clearing the bacteria equally well. From these contrasting results, either other phlogistic factors are operant in the lung or gram-negative and gram-positive bacteria have different requirements for complement-mediated clearance. In terms of human disease, isolated complement deficiencies are very rare, and few patients have been observed. While they do have trouble with infections, these are usually of minor consequence and certainly compatible with survival; the respiratory tract is but one of several troublesome areas. Moreover, this situation points to the efficacy of other host defense systems, which can compensate to overcome a deficiency of another one. 74

Macrophages Lack Proper Cellular Activation or CellMediated Immunity A small group of microbes seem to have adapted for survival in macrophages, and their containment or eradication can be difficult for this phagocyte (Table 2). Obviously, some of these microbes are not involved in lung infections, but they do afflict tissue macrophages in other organ systems. Macrophages are generally considered to be the first line of defense against facultative and obligate intracellular parasites such as M. tuberculosis and M. leprae. Although macrophages can ingest and contain these organisms, unless delayed hypersensitivity and cell-mediated immune functions of the host are operating well, these microbes are not killed but can survive within the cell. For other microbes listed in Table 2, such as Toxoplasma and Legionella pneumophila, macrophages or monocytes are not as effective as PMNs in killing. Pneumocytis carinii also is contained by macrophages in the lungs, but immunosuppressive therapy with corticosteroids can cause it to proliferate in lung tissue. Superimposed on the difficulty that normal lung macrophages may have containing and killing certain microbes, the potential problem of an acquired defect in macrophage and/or lymphocyte functions in the lungs can compound the susceptibility to infection. Recently, this interaction has been dramatized with the vivid appearance of a new epidemic form of lung disease generally found in homosexual men who have antecedent cytomegalovirus (or herpes simplex) respiratory tract infection and develop a fulminant form of Pneumocystis pneumonia TABLE 8.-INTRACELLULARMICROBES CONTAINEDBY (ORRESIDENTIN?) HUMAN MONOCYTES AND MACROPHAGES Mycobacterium tuberculosis Mycobacterium lepraemurium Toxoplasma gondii Leishmania donovani Legionella pneumophila Pneumocystis carinii Listeria monocytogenes Cytomegalovirus 75

or atypical mycobacterial infection. Another example of the macrophage’s need to develop cellular immunity to adequately contain a pathogenic bacterium is provided by legionellosis. Legionella pneumophila is one of the least seven species of Legionella organisms that have been associated with an increasingly diverse spectrum of human disease since the pathogen was first identified, following the well-known outbreak of pneumonia at a state American Legion convention in Philadelphia in July 1976.214-216 Horwitz and Silverstein have studied extensively the roles of antibody, complement, polymorphonuclear leukocytes, and mononuclear cells in host defense against Legionella infection. Legionella pneumophila is a facultative intracellular pathogen that multiplies extensively in peripheral blood monocytes.217 They demonstrated that human serum was ineffective in killing L. pneumophila even in the presence of high titers of anti-Legionella antibody, and that these antibodies were relatively inefficient in fixing complement to the surface of the organism.21s Both antibody and complement were necessary for significant phagocytosis of L. pneumophila by PMNs, yet even under these conditions the organism was relatively resistant to killing by the phagocytes. They concluded that PMNs and the humoral immune system were not decisive components of host defense against Legionella infection. In the presence of anti-L. pneumophila antibody, binding of Legionella organisms to monocytes was enhanced and monocytes, as with PMNs, required both antibody and complement to kill any organisms.‘i’ However, even in the presence of antibody and complement, monocytes killed only a small portion of the Legionella organisms, and intracellular multiplication of surviving organisms was not inhibited. The importance of the monocyte, however, in host responses to Legionella infection was finally demonstrated by showing that activated monocytes could inhibit the intracellular multiplication of L. pneumophila.220 In this study, monocytes from individuals without previous exposure to Legionella infection could be activated by cytokines produced by mitogen-stimulated mononuclear cell cultures. These “activated” monocytes inhibited L. pneumophila 76

multiplication in two ways. First, their phagocytic uptake of organisms was decreased, thus diminishing the numbers of organisms with access to the intracellular environment where the bacteria are known to proliferate.217 Second, the multiplication rate of intracellular L. pneumophila was decreased b the activated monocytes and alveolar macro23J The mechanism by which this inhibition of inphages. tracellular proliferation is accomplished is not yet clear. Taken together, these results underscore the importance of cell-mediated processes, as opposed to bacterial opsonization by antibody and complement, in immune reactions to L. pneumophila. That cell-mediated immunity develops in patients with Legionnaires’ disease is demonstrated by the observation that mononuclear cells from patients with Legionella infection can generate cytokines when incubated with Formalin-killed organisms.221 These cytokines activate monocytes and macrophages, similar to those cytokines released by mitogen-stimulated mononuclear cells, and the cytokine-activated monocytes and alveolar macrophages inhibit intracellular multiplication of the organism. In summary, humoral immunity plays a minor role in host response to L. pneumophila infection, and activation of the mononuclear phagocyte system appears to be of crucial importance for controlling proliferation of this intracellular pathogen. Legionella provides a good contemporary example of the importance of developing delayed cellular immunity to cope optimally with a potentially pathogenic organism. Other comparable situations exist and will be referred to briefly.222 Control of infection with M. tuberculosis, M. leprae, Salmonella typhimurium, Listeria monocytogenes, Toxoplasma gondii, and Leishmania protozoa all require that activated macrophages exist; as a prelude, immune T lymphocytes must develop and through secretion of lymphokines provide cellular mediators that activate the macrophages. Many investigators have studied these lymphocytemacrophage-microbe interactions cited, but the pioneering work of Mackaness223’ 224 and his colleagues more than two decades ago introduced the modern concepts of delayed hypersensitivity and mechanisms of cellular immunity; therefore their work deserves special mention. Lung infection 77

with the protozoan-like organism, P. carinii, is a special case. Pneumocystis lung infection is quite common in immunosuppressed patients, especially those with leukemias and bone marrow failure; infection is like1 to blossom J when doses of corticosteroids are reduced.225’ 26 In certain animals, Pneumocystis organisms seem to be part of the alveolar flora, and treatment with corticosteroids in rats, for example, can induce an infection in almost all of the rodents. Of recent interest has been Pneumocystis infection in subjects with the acquired immunodeficiency syndrome who are also troubled with viral respiratory infections and atypical mycobacterial organisms and some others listed in Table 2. Although data concerning monocyte and macrophage function in AIDS are few at present, the observed propensity for patients with this disorder to have infection with intracellular microbes such as P. carinii, T. gondii, M. avium-intracellulare, and cytomegalovirus suggests that the function of these key host defense cells might be deficient. The respiratory system offers a unique opportunity to study the mononuclear phagocyte system in this disorder. Studies from our laboratory and others have demonstrated that, in contrast to the peripheral T cell lymphopenia observed in AIDS patients, the numbers of the airway T cells obtained b bronchoalveolar lavage are norH mal or increased.227’ 228, 41 There is a striking predominance of OKT8 (monoclonal antiserum) positive suppressor/cytotoxic T lymphocytes in this lung cell population. It is possible that this observed imbalance between the putative T helper and T suppressor cell types causes defects (inadequate lymphokine secretion?) in the ability of alveolar macrophages to become activated, to engulf, and to destroy invading pathogens such as P. carinii. This might account for the increased susceptibility to infection and tumor (Kaposi’s sarcoma) found in AIDS patients. PMNs

ANDTHE LUNG INFLAMMATORY RESPONSE

The complex nature of altered lung host defenses in the heavily immunosuppressed patient is not known completely, especially the function of perhaps minor and inconspicuous components. The impact of cytotoxic chemother78

apy on the regeneration of airway ciliated epithelial cells or on the function of their cilia has not been assessed. Similarly, local secretion along the airways of such immunoglobulins as IgA could be diminished because of sluggishly synthesizing submucosal plasma cells. Damage to other populations of lymphoid cells in the lungs that are involved with cell-mediated immunity has not been systematically assessed, either. However, the effects of granulocytopenia, and hence an impaired inflammatory cell response in the lungs, have been well correlated with susceptibility to a variety of aerobic gram-negative bacteria and fungi (Aspergillus). As listed in Table 1, the ability to mount an appropriate inflammatory response in lung tissue is an important component of host defense. This mechanism is a complex biologic reaction requiring specific initiation, containing steps for amplification that allow systemic elements of immunity to be recruited into the lung (chemotaxins), and, finally, terminating the reaction and allowing subsequent tissue repair. Although several kinds of phagocytic cells participate, PMNs are the most numerous and perhaps the most important host factor once the full-blown inflammatory response has begun. Normal lungs contain PMNs sequestered in interstitial areas and marginated in capillaries. The lungs also have ready access to the circulating granulocyte pool. Previously, in considering various stimuli and chemotactic factors that might attract PMNs to alveoli and airways, we simply assumed that an ample supply of PMNs existed to support the requirements of a local inflammatory reaction. This might not be the case, for either a deficient number of cells or abnormal function can result in an insufficient migration of PMNs into lung tissue and a blunted response. An absolute deficiency of PMNs is not uncommon. The leukopenic patient, often granulocytopenic from antineoplastic chemotherapy or other forms of immunosuppression, has insufficient reserve bone marrow function to support a peripheral white blood cell population and an adequate marginated 001. Infection, particularly bacterial or fungal pneumonia, B” is frequent in these patients and is a common cause of death. The offending organisms are 79

usually Pseudomonas or gram-negative bacilli that inhabit the normal gut but commonly colonize the oropharynx of debilitated hospitalized patients. An inadequate number of circulating PMNs in the blood is one of the best predictors of susceptibility to gram-negative bacillary infection. In lung vasculature, PMNs arrive in the blood and either pass through the capillaries or linger and stick to the endothelium, thus becoming marginated and temporarily stored. Margination is a resting stage for this short-lived phagocytic cell. However, marginated cells remain in dynamic equilibrium with circulating cells, so margination might represent only a temporary removal from the intravascular circuit. Recently, a number of interesting observations have been made regarding PMN adherence to vascular endothelium. Whether the adherence implicit in margination is the same as the “activated” sticking preliminary to the PMN’s egress into lung parenchyma is unknown. However, it is accepted that “sticking” is a prerequisite to PMNs leaving the vascular space and migrating to an extravascular site. Although the mechanisms that cause PMNs to marginate and stick to vascular endothelium have not been precisely defined, a number of conditions that alter adherence or decrease sticking have been investigated. Because sticking seems necessary before PMN diapedesis or egress into tissue can occur, it is a vulnerable step that can interfere with the inflammatory response. A variety of agents and conditions can decrease PMN sticking, including ethanol,230 corticosteroids, and other anti-inflammatory drugs such as aspirin.231 Exocytosis of an appreciable portion of the PMN granule-associated enzymes can effect in vitro adherence and motility.232 With secretion of 30% or more of the lysozyme content, cell adhesiveness was increased and chemotaxis was inhibited, whereas with more limited exocytosis of the intracellular granules’ contents, chemotactic responses were increased. Low levels of magnesium ion in cell-cultured medium also can reduce adhesiveness.233 Thus, events that interfere with the adherence of PMNs to capillary endothelial surfaces might functionally limit the number of phagocytic cells that can be mobilized to an inflamed site. 80

The direct application of such reasoning to clinical problems is tempting but is not yet well substantiated. For example, the inebriated alcoholic who loses consciousness and aspirates oropharyngeal bacteria in the process might develop a pneumococcal or Klebsiella pneumonia. Stupor, stasis, and pooling of respiratory secretions, poor cough response, and other depressed host factors all contribute to establishing the infection. In addition, poor PMN sticking, also attributed to acute ethanol intake, might impair the local inflammatory process in the lung and allow bacteria to proliferate. Occasionally, intrinsic defects in the PMNs can account for poor extravascular migration and susceptibility to infection.240 PMNs from subjects with Chediak-Higashi syndrome, who have large, abnormally fused lysosomes, do not deform well mechanically and therefore respond sluggishly to chemotactic stimuli.’ 4 At least one patient has been found with a deficiency in actin, part of the actin-myosin filamentous structure that propels PMN cytoplasm; the deficiency resulted in poor granulocyte movement.235 Finally, some PMNs exhibit poor spontaneous motility in vitro; they are termed “lazy leukocytes.“236 To review further the multitude of metabolic and biochemical abnormalities that can affect PMNs is not our intention. Although these deficiencies cause general problems with phagocytic intracellular killing and inflammation, they may not produce specific disorders in the lungs. However, the point to emphasize is that a variety of things can impair the response of PMNs in the inflammatory reaction. External factors are important because insufficient chemotactic stimuli might arrive to direct migration. The role of other external factors, such as drugs that impair PMN adherence, is obvious. However, appropriate stimuli might not interact with an appropriately responsive PMN cell-one that is compromised by poor intrinsic mobility or excessive granular enzyme depletion, for example. The net effect is an inadequate influx or concentration of PMN phagocytes in alveoli and parenchyma and an inability of the lung to cope with an infectious inoculum or other troublesome particulate substances or antigens. 81

SUMMARY Serious respiratory tract infections are rare in the healthy individual and most of the nuisance morbidity that occurs results from nasopharyngeal viral infections that many people get once or twice a year. The economic impact from these upper respiratory tract infections is appreciable, however, in terms of absenteeism from school or work, but unfortunately there is little that can be done to ward them off in a practical way. Pneumonia is an infrequent lifetime experience for most non-smoking adults and when it occurs, unusual circumstances may pertain-a particularly virulent microorganism is in circulation, or perhaps one has been exposed to a newly recognized germ, such as has occurred with Legionella species in the past 8 years or so. What protects us the great majority of the time is a very effective network of respiratory tract host defenses. These include many mechanical and anatomical barrier mechanisms concentrated in nose and throat; mucociliary clearance, coughing and mucosal immunoglobulins in the conducting airways and in the air-exchange region of the alveolar structures, phagocytes, opsonins, complement, surfactant and many other factors combine to clear infectious agents. The ability to mount an inflammatory response in the alveoli may represent the maximal and ultimate expression of local host defense. In some way these host defenses are combating constantly the influx of micro-organisms, usually inhaled or aspirated into the airways, that try to gain a foothold on the mucosal surface and colonize it. But many general changes in overall health such as debility, poor nutrition, metabolic derangements, bone marrow suppression and perhaps aging promote abnormal microbial colonization and undermine the body’s defenses that try to cope with the situation. It is a dynamic struggle. The departure from normal respiratory health may not be obvious immediately to the patient or to the physician and repeated episodes of infection or persisting symptoms of cough, expectoration and sinus or ear infections may develop before serious assessment of the situation is taken and appropriate diagnosis gotten underway. Obvious explanations for respiratory infections may be apparent and, 82

nowadays, side effects from antineoplastic chemotherapy or immunosuppressive therapy for a variety of diseases that create an immunocompromised host are common. In a few subjects, especially young adults who present with a cumulative history of frequent but mild infections in childhood and youth, a subtle deficiency in host defenses may exist and have been partially masked because of attentive pediatric medical care and prompt use of broad spectrum antibiotics. Some of these situations have been reviewed here, emphasizing diseases of cilia ultrastructure causing faulty clearance of airway secretions, cystic fibrosis and immunoglobulin deficiencies especially of IgG subclasses and functional destruction of secretory IgA by bacterial proteases. A number of “situational” pneumonias can occur when alveolar macrophages are confronted with intracellular parasites or bacteria that they cannot kill readily unless cellular activation through cell-mediated immunity develops. Such opportunistic infections with Pneumocystis carinii and Legionella pneumophilia highlight the problem, but other related problems with phagocytosis and intracellular killing can result from insufficient opsonic antibodies and lack of other components of humoral and complement cascade mediated immunity. Sometimes the fault lies in generating a vigorous inflammatory reaction. Usually granulocytopenia as part of bone marrow insufficiency is the cause, but other intrinsic defects in polymorphonuclear granulocyte function can be found, too. The message is to be alert and suspect the unexpected when dealing with troublesome respiratory infections that do not fit the norm or have a peculiar natural history. ACKNOWLEDGMENT The author appreciates the secretarial Mae Day and Ms. Joan Pacquette.

assistance

of Mrs.

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SELF-ASSESSMENT 1. a, b, c, d, e

2. a, b, c 3. a, c, d, e 4. a, d, f

98

ANSWERS

5. a, d, e 6. b, c, d 7. a. d