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Inhalation challenge and skin testing in farmer's lung
Inhalation challenge in farmer’s lung J. H. Edwards, Penarth,
and skin testing
Ph.D., and 6. H. Davies,
South Glamorgan,
M.R.C.P.
Wales
An immunologic basis for episodes of farmer’s lung is shown by the response of susceptible individuals to inhalation challenge with extracts of Micropolyspora faeni, the main sensitizing agent in moldy hay dust. However, if the complement-fixing capacity of the farmer’s serum for M. faeni antigen is used as a quantitative assessment of sensitivity, it does not predict whether an individual will respond to inhalation challenge. A positive immediate skin test to M. faeni antigens was observed in all cases with over 40 CHSO consuming units of antibody per milliliter of serum. A similar correlation was seen between complement consumption and precipitins. (J ALLERGY CLIN IMMJNOL 68.38, 1981.)
Inhalation challenge has been a diagnostic test used by allergists since the establishment of extraneous allergens as causative agents in asthma. r Relatively recently this technique has been applied to the extrinsic allergic alveolitis group of diseases, farmer’s lung,2 pigeon breeder’s lung,3 maltworker’s lung,4 and humidifier fever,5 with the use of crude2 or purified6 extracts or whole4 or homogenized microorganisms. ’ Characteristically a response to inhalation challenge in extrinsic allergic alveolitis occurs several hours after exposure and usually consists of a pyrexial episode with a decrease in pulmonary function. The hypothesis that diseases in the extrinsic allergic alveolitis group had an immunologic basis was first suggested from the work of Pepys et al.8 with the demonstration of precipitin antibodies to moldy hay in the serum of patients with farmer’s lung. Later9 it was shown that the precipitins were directed against Micropolyspora faeni, a component of moldy hay dust, and the disease was reproduced in susceptible individuals by inhalation challenge with M. faeni extracts lo as well as moldy hay extracts. * The possibility that such diseases were mediated by immune complexes was suggested by Pepys. ‘I Certainly injurious reactions caused by immune complex formation or deposition have been established in other organs,
From the. Medical Research Council Pneumoconiosis Unit, Llandough Hospital, Penarth, South Glamorgan, Wales, Received for publication July 18, 1980. Accepted for publication April 13, 1981. Reprint requests to: Dr. J. H. Edwards, MRC Pneumoconiosis Unit, Llandough Hospital, Penarth, S. Glamorgan CF6 1XW. Vol.
68, No.
1, pp. 58-64
e.g., acute glomerulonephritis in the kidney,12 or systemically, e.g., serum sickness. Barrowcliff and Arblaster13 considered a fatal acute case of farmer’s lung to be caused by a pulmonary Arthus reaction, I4 i.e., an immune complex reaction. Other investigators have considered different types of hypersensitivity reactions to be important in extrinsic allergic alveolitis, type I reaction,i5 type II reaction,16 or a type IV reaction,” or even in nonspecific complement activation by the alternative pathway6 or specific macrophage stimulation in the absence of antibody. I8 All these components may well play a part, the most influential depending not only on the prevalent type(s) of hypersensitivity but also on the amount of material inhaled and its inherent biologic activity. If, however, tissue damage/disease proceeds by immune complex (type III) pathway, then basically it involves complement-fixing antibodies,ls the ingestion of immune complexes by polymorphonuclear leukocytes and release of hydrolaseszO mediating the tissue damage,21 although there are other complement-dependent features of a type III reaction, e.g. platelet and macrophage activation, that might also contribute to the overall response. Logically the greater the capacity to activate complement of the farmer’s lung serum in combination with antigen, the greater the tissue damage that ensues (passive cutaneous anaphylaxis in guinea pigs22) and presumably the likelihood of an episode of farmer’s lung being produced on challenge; certainly the higher titers/grades of antibody to M. faeni correlate with severity of disease.lO The development of a suitable culture technique for
0091-6749/81/070058+07$00.7010
0
1981
The
C. V. Mosby
Co.
“OLUME WMBER
Inhalation
68 1
TABLE
1. State of 16 cases previously
Name
Age
Duration of disease (yr)
Last acute
diagnosed known episode (yr)
as farmer’s Last contact with hay (yr)
challenge,
skin test in farmer’s
Lung
function
54 26 58
5 3 1%
5 3 None
y3 Constant Constant
M. ‘4. B.
58 35
20 3
2 %
2 Constant
i L. J. I. N. J
3x 38
None None
3 Constant
;,. M. J.
68
16
15
15
‘I- A. R. 0.
I5
4
4
4
1:. H. J.
35
20
None
2
Mild airways obstruction Restriction T,,coi
3. G. L ;. J.
58 50
9 18
9 18
8 8
Slight restriction T,co~ Airways obstruction
3. C. J.
54
11
11
11
i. 0. G.
65
40
3
I
._ J.
35
12
12
3
3. -
55
1
‘h
R.J
Slight restriction Normal Airways obstruction T,.d Obstructive pattern Normal Normal Airways obstruction (as1hma) Restriction T,,coL
Airways obstruction T,~(‘o i Airways obstruction T,.coi Normal
Constant
59
lung
). R. G i G. J. : P
Asymptomatic 3
lung
Obstructive
pattern
Cheat
X-ray
Fine diffuse nodularity Nomlal Empbksema. irregular shadows mid zones Emphysema Increaschd nodules upper IOI’C\ Normal Hyperinflatcd Emphysema: fibrosis Normal
upper lobe
Fine nodular upper lobe infiltrate Hyperinflaled Emphysema; upper lobe fibrosis Emphysema; upper lobe fibrosis Emphycema: fine nodular change Irregular nodular infilWale upper zones Emphysema
T,co = transfer factor of the lungs for carbon monoxide. M. faeni antigens23 has allowed their for both inhalation challenge and skin testing,‘j whereas previously, skin tests with moldy hay extracts produced irritant reactions” or ambiguous results. * Thus this study was undertaken to determine whether the complement-consuming capacity of farmer’s lung serum for M. fueni antigens correlated with and could predict an episode of farmer’s lung after inhalation challenge with low doses of M. faeni antigen. It was not our intention to use a high dose of M. ,faeni antigen because if all of the past cases responded to challenge it would then be impossible to establish a correlation between complement activation and response. Rather, a low dose sufficient to produce a response in only a fraction of the subjects was considered more correct for this type of study. Similarly, a correlation between skin-test reactivity and potential response to challenge was sought. harvesting use
MATERlALS AND METHODS Farmer’s lung patients Patients entering into the study were referred to us by local physicians as past cases of farmer’s lung. They were diagnosed as farmer’s lung patients on the basis
of history. chest radiologic examination.
and precipitins against M. ,fhrni. Histop. Twelve of the 16 past cases exhibited symptoms of breathlessness, cough, malaise, and fever occurring 4 to 12 hr after Iexposure to moldy hay dust on at least two occasions, and for these it was possible to estimate the date of the last acute attack; this ranged from 6 mo to 18 yr. Patient E. L. J. had no history of acute farmer’s lung but had transient pulmonary infiltrates and positive precipitins at the time of original diagnosis. Chesremminution. Nine of the 16 past caseshad persisting abnormalities of upper lobe pulmonary fibrosis or diffuse nodularity. Two of the 16 had an emphysematous pattern, two were hyperinflated, and three were radiologically normal. Serology. Eleven of the 16 had current precipitins against M. fueni anligens. Their clinical states, radiographic involvement, and present exposure patterns are reviewed in Table I. The night before challnge each individual was skin tested intradermally with a.pproximately 10 to 20 ~1 of purified glycopeptide and prolein antigens of M. ,farni at LOO*g/ml in saline, with a saline control. This was primarily undertaken to ensure that no severe adverse reaction would occur on inhalation challenge, but it also allowed a 36-hr reading to be undertaken before discharge. The next day skin tests were
60
Edwards
and
repeated on the other arm. and recordings were made of the response up to 8 hr after testing. Control skin tests were performed with the sameM. fat~i antigens on 17 patients with other pulmonary disease,e.g., asthma, allergic bronchopulmonary aspergillosis, and pigeon breeder’s lung, as well as on five normal individuals. All results in these groups were negative. M. faeni antigens M. faeni antigens were prepared by the double-dialysis technique of Edwards.23This method allows the preparation of microbial extracts free from any substrate on which the organism has developed. Antigens were used at 5 mgiml for gel diffusion testing and at concentrations up to 10 mg/ml for complement consumption studies. At this highest concentration antigens prepared by the double-dialysis technique do not activate the alternative pathway of complement.“’ For skin testing the antigens were processedfurther, as previously described.2-1Protein antigens were purified by a combination of ethyl alcohol precipitation and chromatography on Sephadex G200 and diethylaminoethyl (DEAE) cellulose.*’ Glycopeptide antigens were purified by a combination of ethyl alcohol, trichloroacetic acid precipitation, and chromatography on G200 and DEAE.2-l Inhalation challenge Inhalation challenge was conducted with a Wright Nebulizer at 1.1 kg/cm2air pressurethat nebulized approximately 2 ml of M. faeni antigen at 1.Omg/ml over a period of 10 min. The nebulized extract was delivered to the individual by a loose-fitting face mask. Temperature was recorded every hour, and the following lung function tests were performed: spirometry, flow-volume curves, lung volumes, and diffusing capacity for carbon monoxide (single breath technique) assessedprior to challenge and then 30 min, 1, 2, 4, 6, and 12 hr after challenge. Immunologic
J. ALLERGY CLIN. IMMUNOL. JULY 1981
Davies
studies
Blood sampleswere taken on the night before challenge, just prior to challenge, and at 2-hr intervals up to 6 hr, then on the following day. All sera were separated and analyzed for total comp1ement.2j The first serum sample was also used for complement-consuming capacity in the region of equivalence with M. faeni antigen. In essence,10 ~1 of serum was added to 10 ~1 aliquots of M. fueni antigen at 1, 10, 100 pug/ml, and 1 and 10 mgiml plus 150 ~1 guinea pig serum. After 1 hr at 37” C, residual complement was titrated as for total complement, and the amount consumed by the antigen antibody reaction was expressed in CH50 units.” For two patients who responded to challenge (D. R. G. and E.P.) measurements of the complement-consuming capacity of each 2-hr sample were undertaken. Skin test biopsy
material
To assessthe histologic response occurring during the immediate skin reaction, arrangements were made to skin
test 10 other patients with farmer’s lung, who were not included in the challenge program. These were provided by Dr. A. Axford, Machynelleth Chest Clinic. Biopsieswere performed 10 min after intradermal inoculation on four of those patients who reacted, and tissue was excised and placed in 10% neutral formalin; one volunteer also had a skin biopsy at 2% hr. After hematoxylin and eosin staining, the biopsieswere examined by Dr. R. M. E. Seal, Llandough Hospital. RESULTS Clinical and physiologic challenge
responses
to
Three patients reacted to inhalation challenge with the development of a pyrexial episode starting at approximately 6 hr, peaking at 15 hr, and gradually resolving at 20 br after challenge. Patients described a general feeling of malaise, and each subject likened his induced episode to that experienced after handling moldy hay. In all three there was a fall in vital capacity (VC) and forced expiratory volume in one second (FEV,), and a delayed pattern was seen at 12 hr (Table II). One other patient (A. N. J.) produced an immediate response with a fall in FEV, and VC at 30 min, which we consider to be caused by his asthmatic condition. Response to challenge complement-consuming
vs capacity
It can be seen that the response to challenge is not “predicted” by the complement-consuming capacity of the serum for M. fueni antigen (p > 0.1, MannWhitney U test). This was most marked in two patients (Table II): D. R. G. responded to challenge with a complement-consuming capacity of 40 CH50 units/ml of serum, whereas E. G. L., with a complement-consuming capacity of 1630 CH50 units/ ml of serum, failed to respond to challenge. Ten of the 16 patients had values between 40 and 1600 CH50 units/ml of serum, and of these two (E. R. J. and E. P.) reacted with a complement-consuming capacity of 750 and 710 CH50 units/ml, respectively. Interestingly, patient E. P. responded to a very small dose of antigen (700 pg), and considering that not all the nebulized material reached the lungs by virtue of condensation and expiration, it shows that for some highly sensitized individuals an extremely small dose can provoke a reaction. Changes levels
in complement
levels and antibody
There were no significant increases or decreases in CH50 levels over the first 6-hr period after challenge for either responding patients (two tested) or nonresponding patients (12 tested) (Fig. 1). Moreover,
VOLllME 68 NUVBER I
TABLE -_. --
inhalation
II. Response
to
inhalation
challenge
with
M. faeni
purified
challenge,
skin
KH50) 40
2 mg
E. :; J E. I’.
1s 7 IO
2mg 700 pg
I. ‘,I H. 3 E. I. J. A. \.J
‘OS 243 56 IS
1 1 I.2 1.3
w mg mg mg
+ Starting at 6 hr, peak 14 hr Mild at 6-hr. severe 15-hr -
0 Y 85 1630 34
2.3 1.7 2.2 2 2.1
mg mg
-
mg
-
+
++--
-
mg
-
+
++--
-
mg
-
2 mg
-
+
++--
-
+ +
++-+---
+ Starting at 5 hr, lasting
+
+
E. T. C. E. F.
Name 1 c-i.
!“I. J. ‘\. R. 0. -1. J ci. L. i.
11. .‘. J.
54
Ii. 0,
G.
80
L. ,. E. I< J
66 750
2.8 mg 2 mg 2w
Response
test
10 min
2% hr
6 hr
24 hr
36 hr
+
-
-
-
-
-
= negative;
MEFR
= maximum
expiratory
+
+ + +
++-++-++--
i
-
--
-
Fall in spirometry 6i MEFR
+
-
61
- Fall in spirometry (12 hr) -- Immediate asthmatic reaction -. -
-
-----_____
Maximum temperetures (“Cl
Precfpitins against M. faeni
39.5 at i 2 hr
4 +
39.8 at I? hr
+ +-k
hr)
++ 4
t+
*+
c
+ c+ +
None but cough & dyspnea present -
-
- Fall in FEV,, in- 38.3 at X hr crease in obstructive
pattern
t + ii+
---
Row rate.
there was no decline in complement-fixing antibody in two of the responding patients (D. R. G. and E. P.) (Table III). All sera with values of >30 CH50 units/ml produced a positive precipitin response. This is ?Jatistically significant (p < 0.01). Skin test results
Lung function after challenge
(I2
13 hr
-_. + .: positive;
Skin
+
lung
extract
Challenge dose of M. faeni extract
D.
in farmer’s
--. Complement consumption maximum
--.
test
and biopsies
The skin-test results were similar to those experienced previously. fi For the challenged patients, 11 of the 16 produced an immediate skin reaction of central edema, sometimes with pseudopods and surrounding er) thematous zone between 2 and 10 min. The edematcus response was up to 3 by 2 cm in area (patient D R. G .). The skin reaction resolved in one patient at 211: hr, but for eight of the 11 the maximal time of inrvolvement was 2 to 3 hr; thereafter the reaction pelsked at 6 hr. All skin tests were negative or had re!.idual involvement only at 24 and 36 hr. There were only marginal qualitative differences between the reactions to proteins or glycopeptide antigens. For the recently diagnosed patients, whose skin tests were observed only up to 4 hr (excluding two patients on stczroids), six of eight were positive at 15 min, five of se ien were positive at 1 hr, three of four were positive
at 2 hr, and two of three were positive at 4 hr. Three biopsies performed at 10 min revealed edema as the main feature; in only one biopsy was diapedesis of eosinophils seen in several small vessels. An interesting correlation was observed (Table II) between positive immediate skin-test responses and complement-consuming capacities of 40 or more CH50 units/ml of serum (p = 0.0002). DtSCUSSlON
The results show that the complement-consuming ability of farmer’s lung serum does not forecast the capacity of an individual to respond to inhalation challenge using relatively low doses of M. ,faeni antigen with an episode of farmer’s lung. Although this might have been predicted from the presence of healthy farmers with precipitins against M. faeni in a farming population, 26 it was possible that the&seantibodies were of the poorly or non-complement-fixing subgroups of IgG (i.e., IgG2 and IgG4). Our results do not appear to confirm those of de Ridder and Berrens,27 who were able to differentiate between precipitin-positive asymptomatic pigeon breeders and symptomatic cases of pigeon breeder’s
52
J. ALLERGY CLIN. IMMUNOL. JULY 1981
Edwards and Davies
3
2
1
h rs.
after
4
exposure.
1. Complement levels after challenge. Responding no general trend in CH50 levels for either responding
FIG.
TABLE III. Complement consumption per milliliter serum of two responding challenge patients with M. feani antigen at zone of equivalence (1 mg/ml)* Time
after
challenge (hrl
D. R. G.
E. P.
Start 2 4 6 I4 24
40 47.9 40.5 ND 44.4 43.6
710 730 760 775 ND 775
ND = not done. *Note no fall in antibodies during
24 hr after
capable
of complement
consumption
challenge.
lung on the basis of complement-consumption tests. Moore et a1.28 also found generally higher titers of complement-fixing antibodies in symptomatic pigeon breeders. However, the work presented here and our relevant inhalation challenge studies in humidifier fever2s indicate that in these two diseases patients who respond to challenge do not necessarily have higher titers of complement-consuming antibodies than nonresponders. Perhaps pigeon breeder’s lung differs in
5
6
14 Fig. la.
patients are asterisked. Note that there is or non-responding challenge patients.
this respect from farmer’s lung and humidifier fever; indeed, the failure of M. fueni and humidifier fever antigens29 to reduce complement levels in both responders and non-responders differs from the results of Moore et a1.28These authors found that complement levels of precipitin-positive asymptomatic pigeon breeders fell after challenge, whereas the complement levels of symptomatic cases were not affected. We have not evaluated the immunoglobulin(s) responsible for the immediate skin reactions seen in 11 of the 16 patients, but Patterson et a1.30 have shown that IgE antibody against M. faeni was not a feature of the farmer’s lung cases they studied. There is therefore the possibility that the immediate skin reactions were caused by IgG4 involvement.31 What then is the nature of the response to the inhaled material? It is unlikely to be alternative pathway complement activation, since M. fueni extracts prepared in our laboratory did not activate the alternative pathway even at ten-times the concentration used.32 Also, direct stimulation of macrophagesLB is unlikely, since one would expect a more uniform response to the challenge doses if this were so. If complement-fixing antibodies in lung secretions are the same as those in circulation, then correlation might be expected between complement-consuming
VOL. JME 68 NUlilBER 1
capacity and response for immune complex-induced epliodes. Deuschl and Johansson33 found an average of 8% IgG to be produced locally in the lungs of normal individuals, but the percentage varied greatly when groups with known bronchopulmonary disease were tesed; figures for extrinsic allergic alveolitis were not inciuded, and IgM levels were not determined. ‘The time course of the responses was not that of an lgE,-mediated reaction. Although 11 of 16 patients ga*‘e immediate skin test responses, in only one of thei;e was there evidence of a decrease in pulmonary fuI!ction within the first 30 min after challenge. We car postulate a cell-mediated immune response, and Hansen and Penny3” consider cell-mediated immunity to ‘)e a feature of pigeon breeder’s lung, but the skin reactions observed were not of the delayed type. However, skin reactions may not reflect the immunologic capacity and/or responsiveness of the lung, considering the observation of negative skin tests but po!,itive response to nasal challenge with mite extrat:ts.35 The time course of a delayed reaction (18+ hr n skin) may well be shortened in the lung because of the relatively greater numbers of lymphocytes in situ or derived from the circulation. We have shown pu monary responses to purified protein derivative of M:I cobacterium tuberculosis (BCG) in BCG-immunixed rabbits at 3 and 9 hr,36 but this was not the experience of Richerson with aerosol challenge of tht; protein derivative in guinea pigs sensitized with Frt:und’s complete adjuvant (containing killed M. tubcwulosis organisms), where at 6 hr there were only mElima lesions. Perhaps the difference was in the do”;e administered. 4n increasing number of laboratories emphasize the role of the lung as an almost independent organ immunologically. Hunninghake and Fauci38 have shown that 10” sheep red blood cells introduced intrauacheally into guinea pig lungs produced plaqueforming cells (complement-fixing antibody-producing ceils) in the hilar lymph node. At 10 X log sheep red blrlod cells introduced intratracheally, plaque-forming ceils were found in lungs, hilar lymph nodes, and to a IeFser extent the spleen, whereas with 20 X log sheep red blood cells the spleen became the major organ for phque-forming cells of the lungs, hilar lymph node, an 1 spleen, There seems to be a critical dose above which antigenic stimuli spill over into the circulation, anl this may involve active transport of antigens (or bn:akdown products) by macrophages to other lymphoid tissue. For farmer’s lung the inhaled material is mainly particulate, 39 and our work on the respirable fraction of moldy hay shows that the soluble material in respirable moldy hay dust, although capable of activat-
Inhalation
challenge,
skin test in farmer’s
lung
63
ing Cl directly,“” does not contain antigens of M. fueni as judged by the failure of concentrated extracts of the soluble material to react with farmer’s lung serum. The particulate material contains antigens that absorb out antibodies to the cell wall of M. @eni (unpublished observationj. Thus the antigenic components of moldy hay dust, since they are not soluble and diffusable, are trapped in the lungs, and experimentally the macrophage is the initial phagocytic cell.-‘O
There are therefore good reasons for the development of local immunity, certainly in farmer’s lung. Subsequent inhalation challenge would then involve immunoglobulins and cells present within the lung and might not necessarily be reflected in the circulation, particularly IgA and 1gE.I”’ Evidence for local pulmonary immunity has been provided experimentally by Kawai et a6., ii with use of bronchial la.vage cells from rabbits immunized intratracheally with thermophilic actinomycete antigen, and in human disease by Schuyler et ai. .I1 who demonstrated macrophage migration inhibitory factor to be produced by bronchial lavage cells from a patient with pigeon breeder’s lung incubated with pigeon serum or pigeon droppings extract. Peripheral lymphocytes did not produce migration inhibition factor under the same conditions. When challenged with pigeon serum the patient responded with an attack of pigeon breeder’s lung, and afterwards the bronchial lavage cells were refractory for up to 7 days. The refractory penod was noticed by Williams? in his classic study of inhalation challenge in farmer’s lung with
moldy
hay extracts
and may be the result
of
several factors such as persistence of antigen in the lung? The depletion of intracellular biologically active factors could also cause a refractory period, which would fit well with our observations on humidifier fever,4’:’ where pyrexial episodes occurred on the first day back to work after a break; a build-up of factors such as lymphocyte pyrogen Factor-” had occurred. It therefore seems that local immunity m the lung provides many answers to the problems encountered in extrinsic allergic alveolitis, and studies employing bronchiolar lavage technique should provide solutions to some of these outstanding problems We expressour appreciation for the help given by Drs. G. 0. Thomas and A. Axford, Machynlleth Chest Clinic. Dr. G. 0. Thomas, Neville Hall Hospital. Abergavenny. and Dr. D. J. Jones, Withybush Hospital. Ilaverfordwest. REFERENCES
1. Ramazzini B: De morbus artificum. Chicago. 1940. University of Chicago Press.
64
Edwards
and
J. ALLERGY CLIN. IMMUNOL. JULY 1981
Davies
2. Williams JV: Inhalation and skin tests with extracts of hay and fungi in patients with farmer’s lung. Thorax 18:182, 1963. 3. Reed CE, Sosman A, Barbee RA: Pigeon breeders’ lung. JAMA 193:81,, 1965. 4. Riddle HF, Channel1 S, Blyth W, Weir DM, Lloyd M, Amos WMG, Grant IWB: Allergic alveolitis in a maltworker. Thorax 23:271, 1968. 5. Amow PM, Fink JN, Schleuter DP, Barboriak JJ, Mallison G, Said SI, Martin S, Unger GF, Scanlon GT, Kurup VP: Early detection of hypersensitivity pneumonitis in office workers. Am J Med 64:236, 1978. 6. Edwards JH, Baker JT, Davies BH: Precipitin test negative farmer’s lung: activation of the alternative pathway of complement by mouldy hay dusts. Clin Allergy 4:379, 1974. 7. Banaszak EF, Thiede WH, Fink JN: Hypersensitivity pneumonitis due to contamination of an air conditioner. N Engl J Med 283:271, 1970. 8. Pepys J, Riddell RW, Citron KM, Clayton YM: Precipitins against extracts of hay and fungi in the serum of patients with farmer’s lung. Acta Allergol 16:76, 1961. 9. Pepys J, Jenkins PA, Festenstein GN, Gregory PH, Lacey ME, Skinner FA: Farmer’s lung, thermophilic actinomycetes as a source of “farmer’s lung hay” antigen. Lancet 2:607, 1963. 10. Pepys J, Jenkins PA: Precipitin (FLH) test in farmer’s lung. Thorax 2Or21, 196.5. 11. Pepys J: Monographs on allergy. Basel, 1969, S. Karger. 12. Teisberg P, Fauchald P, Flatmark A, Myhm E, Mellbye DJ, Jansen H, Brodwall E: Serial complement studies in a patient with Goodpasture’s Syndrome treated with bilateral nephrectomy and renal transplantation. Am J Med 59563, 1975. 13. Barrowcliff DF, Arblaster PG: Farmer’s lung: a study of an early acute fatal case. Thorax 23:490, 1968. 14. Arthus M: Injections rep&es de serum du cheval chez le lapin. Compt Rend Sot Biol 55:871, 1903. 15. Ghose T, Landrigan P, Killen R, Dill J: Immunopathological studies in patients with farmer’s lung. Clin Allergy 4:119, 1974. 16. Wenzel F, Emanual DA, Gray RL: Immunofluorescent studies in patients with farmer’s lung. J ALLERGY CLIN IMMUNOL 4Br224, 1971. 17. Kawai T, Salvaggio J, Harris JO, Arquembourg P: Alveolar macrophage migration inhibition in animals immunized with thermophilic actinomycete antigen. Clin Exp Immunol 15: 123, 1973. 18. Schorlemmer HU, Edwards JH, Davies P, Allison AC: Macrophage responses to mouldy hay dust, Micropolyspora fueni and zymosan, activators of complement by the alternative pathway. Clin Exp Immunol 27:198, 1977. 19. Gel1 PGM, Coombs RAA, editors: Clinical aspects of immunology. Oxford, 1963, Blackwell Scientific Publications, Ltd. 20. Weissman G, Zurier RB, Spieler PJ, Goldstein IM: Mechanisms of lysosomal enzyme release from leucocytes exposed to immune complexes and other particles. J Exp Med 134: 149S, 1971. 21. Hawkins D: Neutrophilic leukocytes in immunologic reactions: evidence for the selective release of lysosomal constituents. J Immunol 108:310, 1972. 22. Ovary 2, Bier OG: Quantitative studies on passive cutaneous anaphylaxis in the guinea pig and its relationship to the Arthus phenomenon. J Immunol 71:6, 1953. 23. Edwards JH: The double dialysis method for producing farmer’s lung antigens. J Lab Clin Med 79:683, 1972.
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Edwards JH: The isolation of antigens associated with farmer’s lung. Clin Exp lmmunol 11:341, 1972. Kabat EA, Mayer MM: Experimental immunochemistry. Springfield, Ill., 1961, Charles C Thomas, Publisher. Grant IWB, Blyth W, Wardrop VE, Gordon RM, Pearson JCG, Mair A: Prevalence of farmer’s lung in Scotland: a pilot survey. Br Med J 1:530, 1972. de Ridder G, Berrens L: Complement consumption in the diagnosis of pigeon breeder’s disease. Stand J Respir Dis 60:243, 1979. Moore VL, Fink JN, Barboriak JJ, Ruff LL, Schlueter DP: Immunologic events in pigeon breeders’ disease. J ALLERGY CLIN ~IUNOL 53:319, 1974. Edwards JH, Cockcroft A: Inhalation challenge in humidifier fever. Clin Allergy. (In press.) Patterson R, Roberts M, Roberts RC, Emanuel DA, Fink JN: Antibodies of different immunoglobulin classes against antigens causing farmer’s lung. Am Rev Respir Dis 114:315, 1976. Vijay HM, Perelmutter L: Inhibition of reagin-mediated PCA reactions in monkeys and histamine release from human leukocytes by human IgG4 subclass. Int Arch Allergy Appl Immunol 53:78, 1977. Edwards JH: A quantitative study on the activation of the alternative pathway of complement by mouldy hay dust and thermophilic actinomycetes. Clin Allergy 6:19, 1976. Deuschl H, Johansson SGG: Immunoglobulins in tracheobronchial secretion with special reference to IgE. Clin Exp Immunol 16:401, 1974. Hansen PJ, Penny R: Pigeon-breeder’s disease. Study of the cell mediated immune response to pigeon antigens by the lymphocyte culture technique. Int Arch Allergy 47:498, 1974. Huggins KG, Brostoff J: Local production of specific IgE antibodies in allergic rhinitis patients with negative skin tests. Lancet 2: 148, 1975. Edwards JH: An investigation of the antigenic complexes associated with farmer’s lung and allied diseases and their pathogenicity. University of Wales, 1968, Ph.D. Thesis. Richerson HB: Acute experimental hypersensitivity pneumonitis in the guinea pig. J Lab Clin Med 79:745, 1972. Hunninghake GW, Fauci AS: Immunologic reactivity of the lung. V. Regulatory effects of antibody on the pulmonary immune response to locally administered antigen. J Immunol 118:1728, 1977. Edwards JH: The effect of soluble material from mouldy hay dust on the complement system. Immunol 37:91, 1979. Edwards JH. Wagner JC, Seal RME: Pulmonary responses to particulate materials capable of activating the alternative pathway of complement. Clin Allergy 6:155, 1976. Schuyler MR, Thigpen TP, Salvaggio J: Local pulmonary immunity in pigeon breeder’s disease. Ann Int Med 88:355, 1978. Salvaggio J, Phanuphak P, Stanford R, Bite D, Claman H: Experimental production of granulomatous pneumonitis. J ALLERGY CLIN IMMUNOL 56:364, 1975. Edwards JH: Microbial and immunological investigations following an outbreak of humidifier fever and remedial action. Br J Ind Med 57:35, 1980. Chao P, Francis L, Atkins E: The release of endogenous pyrogen from guinea pig leukocytes in vitro. J Exp Med 145:1288, 1977.
and skin testing
Ph.D., and 6. H. Davies,
South Glamorgan,
M.R.C.P.
Wales
An immunologic basis for episodes of farmer’s lung is shown by the response of susceptible individuals to inhalation challenge with extracts of Micropolyspora faeni, the main sensitizing agent in moldy hay dust. However, if the complement-fixing capacity of the farmer’s serum for M. faeni antigen is used as a quantitative assessment of sensitivity, it does not predict whether an individual will respond to inhalation challenge. A positive immediate skin test to M. faeni antigens was observed in all cases with over 40 CHSO consuming units of antibody per milliliter of serum. A similar correlation was seen between complement consumption and precipitins. (J ALLERGY CLIN IMMJNOL 68.38, 1981.)
Inhalation challenge has been a diagnostic test used by allergists since the establishment of extraneous allergens as causative agents in asthma. r Relatively recently this technique has been applied to the extrinsic allergic alveolitis group of diseases, farmer’s lung,2 pigeon breeder’s lung,3 maltworker’s lung,4 and humidifier fever,5 with the use of crude2 or purified6 extracts or whole4 or homogenized microorganisms. ’ Characteristically a response to inhalation challenge in extrinsic allergic alveolitis occurs several hours after exposure and usually consists of a pyrexial episode with a decrease in pulmonary function. The hypothesis that diseases in the extrinsic allergic alveolitis group had an immunologic basis was first suggested from the work of Pepys et al.8 with the demonstration of precipitin antibodies to moldy hay in the serum of patients with farmer’s lung. Later9 it was shown that the precipitins were directed against Micropolyspora faeni, a component of moldy hay dust, and the disease was reproduced in susceptible individuals by inhalation challenge with M. faeni extracts lo as well as moldy hay extracts. * The possibility that such diseases were mediated by immune complexes was suggested by Pepys. ‘I Certainly injurious reactions caused by immune complex formation or deposition have been established in other organs,
From the. Medical Research Council Pneumoconiosis Unit, Llandough Hospital, Penarth, South Glamorgan, Wales, Received for publication July 18, 1980. Accepted for publication April 13, 1981. Reprint requests to: Dr. J. H. Edwards, MRC Pneumoconiosis Unit, Llandough Hospital, Penarth, S. Glamorgan CF6 1XW. Vol.
68, No.
1, pp. 58-64
e.g., acute glomerulonephritis in the kidney,12 or systemically, e.g., serum sickness. Barrowcliff and Arblaster13 considered a fatal acute case of farmer’s lung to be caused by a pulmonary Arthus reaction, I4 i.e., an immune complex reaction. Other investigators have considered different types of hypersensitivity reactions to be important in extrinsic allergic alveolitis, type I reaction,i5 type II reaction,16 or a type IV reaction,” or even in nonspecific complement activation by the alternative pathway6 or specific macrophage stimulation in the absence of antibody. I8 All these components may well play a part, the most influential depending not only on the prevalent type(s) of hypersensitivity but also on the amount of material inhaled and its inherent biologic activity. If, however, tissue damage/disease proceeds by immune complex (type III) pathway, then basically it involves complement-fixing antibodies,ls the ingestion of immune complexes by polymorphonuclear leukocytes and release of hydrolaseszO mediating the tissue damage,21 although there are other complement-dependent features of a type III reaction, e.g. platelet and macrophage activation, that might also contribute to the overall response. Logically the greater the capacity to activate complement of the farmer’s lung serum in combination with antigen, the greater the tissue damage that ensues (passive cutaneous anaphylaxis in guinea pigs22) and presumably the likelihood of an episode of farmer’s lung being produced on challenge; certainly the higher titers/grades of antibody to M. faeni correlate with severity of disease.lO The development of a suitable culture technique for
0091-6749/81/070058+07$00.7010
0
1981
The
C. V. Mosby
Co.
“OLUME WMBER
Inhalation
68 1
TABLE
1. State of 16 cases previously
Name
Age
Duration of disease (yr)
Last acute
diagnosed known episode (yr)
as farmer’s Last contact with hay (yr)
challenge,
skin test in farmer’s
Lung
function
54 26 58
5 3 1%
5 3 None
y3 Constant Constant
M. ‘4. B.
58 35
20 3
2 %
2 Constant
i L. J. I. N. J
3x 38
None None
3 Constant
;,. M. J.
68
16
15
15
‘I- A. R. 0.
I5
4
4
4
1:. H. J.
35
20
None
2
Mild airways obstruction Restriction T,,coi
3. G. L ;. J.
58 50
9 18
9 18
8 8
Slight restriction T,co~ Airways obstruction
3. C. J.
54
11
11
11
i. 0. G.
65
40
3
I
._ J.
35
12
12
3
3. -
55
1
‘h
R.J
Slight restriction Normal Airways obstruction T,.d Obstructive pattern Normal Normal Airways obstruction (as1hma) Restriction T,,coL
Airways obstruction T,~(‘o i Airways obstruction T,.coi Normal
Constant
59
lung
). R. G i G. J. : P
Asymptomatic 3
lung
Obstructive
pattern
Cheat
X-ray
Fine diffuse nodularity Nomlal Empbksema. irregular shadows mid zones Emphysema Increaschd nodules upper IOI’C\ Normal Hyperinflatcd Emphysema: fibrosis Normal
upper lobe
Fine nodular upper lobe infiltrate Hyperinflaled Emphysema; upper lobe fibrosis Emphysema; upper lobe fibrosis Emphycema: fine nodular change Irregular nodular infilWale upper zones Emphysema
T,co = transfer factor of the lungs for carbon monoxide. M. faeni antigens23 has allowed their for both inhalation challenge and skin testing,‘j whereas previously, skin tests with moldy hay extracts produced irritant reactions” or ambiguous results. * Thus this study was undertaken to determine whether the complement-consuming capacity of farmer’s lung serum for M. fueni antigens correlated with and could predict an episode of farmer’s lung after inhalation challenge with low doses of M. faeni antigen. It was not our intention to use a high dose of M. ,faeni antigen because if all of the past cases responded to challenge it would then be impossible to establish a correlation between complement activation and response. Rather, a low dose sufficient to produce a response in only a fraction of the subjects was considered more correct for this type of study. Similarly, a correlation between skin-test reactivity and potential response to challenge was sought. harvesting use
MATERlALS AND METHODS Farmer’s lung patients Patients entering into the study were referred to us by local physicians as past cases of farmer’s lung. They were diagnosed as farmer’s lung patients on the basis
of history. chest radiologic examination.
and precipitins against M. ,fhrni. Histop. Twelve of the 16 past cases exhibited symptoms of breathlessness, cough, malaise, and fever occurring 4 to 12 hr after Iexposure to moldy hay dust on at least two occasions, and for these it was possible to estimate the date of the last acute attack; this ranged from 6 mo to 18 yr. Patient E. L. J. had no history of acute farmer’s lung but had transient pulmonary infiltrates and positive precipitins at the time of original diagnosis. Chesremminution. Nine of the 16 past caseshad persisting abnormalities of upper lobe pulmonary fibrosis or diffuse nodularity. Two of the 16 had an emphysematous pattern, two were hyperinflated, and three were radiologically normal. Serology. Eleven of the 16 had current precipitins against M. fueni anligens. Their clinical states, radiographic involvement, and present exposure patterns are reviewed in Table I. The night before challnge each individual was skin tested intradermally with a.pproximately 10 to 20 ~1 of purified glycopeptide and prolein antigens of M. ,farni at LOO*g/ml in saline, with a saline control. This was primarily undertaken to ensure that no severe adverse reaction would occur on inhalation challenge, but it also allowed a 36-hr reading to be undertaken before discharge. The next day skin tests were
60
Edwards
and
repeated on the other arm. and recordings were made of the response up to 8 hr after testing. Control skin tests were performed with the sameM. fat~i antigens on 17 patients with other pulmonary disease,e.g., asthma, allergic bronchopulmonary aspergillosis, and pigeon breeder’s lung, as well as on five normal individuals. All results in these groups were negative. M. faeni antigens M. faeni antigens were prepared by the double-dialysis technique of Edwards.23This method allows the preparation of microbial extracts free from any substrate on which the organism has developed. Antigens were used at 5 mgiml for gel diffusion testing and at concentrations up to 10 mg/ml for complement consumption studies. At this highest concentration antigens prepared by the double-dialysis technique do not activate the alternative pathway of complement.“’ For skin testing the antigens were processedfurther, as previously described.2-1Protein antigens were purified by a combination of ethyl alcohol precipitation and chromatography on Sephadex G200 and diethylaminoethyl (DEAE) cellulose.*’ Glycopeptide antigens were purified by a combination of ethyl alcohol, trichloroacetic acid precipitation, and chromatography on G200 and DEAE.2-l Inhalation challenge Inhalation challenge was conducted with a Wright Nebulizer at 1.1 kg/cm2air pressurethat nebulized approximately 2 ml of M. faeni antigen at 1.Omg/ml over a period of 10 min. The nebulized extract was delivered to the individual by a loose-fitting face mask. Temperature was recorded every hour, and the following lung function tests were performed: spirometry, flow-volume curves, lung volumes, and diffusing capacity for carbon monoxide (single breath technique) assessedprior to challenge and then 30 min, 1, 2, 4, 6, and 12 hr after challenge. Immunologic
J. ALLERGY CLIN. IMMUNOL. JULY 1981
Davies
studies
Blood sampleswere taken on the night before challenge, just prior to challenge, and at 2-hr intervals up to 6 hr, then on the following day. All sera were separated and analyzed for total comp1ement.2j The first serum sample was also used for complement-consuming capacity in the region of equivalence with M. faeni antigen. In essence,10 ~1 of serum was added to 10 ~1 aliquots of M. fueni antigen at 1, 10, 100 pug/ml, and 1 and 10 mgiml plus 150 ~1 guinea pig serum. After 1 hr at 37” C, residual complement was titrated as for total complement, and the amount consumed by the antigen antibody reaction was expressed in CH50 units.” For two patients who responded to challenge (D. R. G. and E.P.) measurements of the complement-consuming capacity of each 2-hr sample were undertaken. Skin test biopsy
material
To assessthe histologic response occurring during the immediate skin reaction, arrangements were made to skin
test 10 other patients with farmer’s lung, who were not included in the challenge program. These were provided by Dr. A. Axford, Machynelleth Chest Clinic. Biopsieswere performed 10 min after intradermal inoculation on four of those patients who reacted, and tissue was excised and placed in 10% neutral formalin; one volunteer also had a skin biopsy at 2% hr. After hematoxylin and eosin staining, the biopsieswere examined by Dr. R. M. E. Seal, Llandough Hospital. RESULTS Clinical and physiologic challenge
responses
to
Three patients reacted to inhalation challenge with the development of a pyrexial episode starting at approximately 6 hr, peaking at 15 hr, and gradually resolving at 20 br after challenge. Patients described a general feeling of malaise, and each subject likened his induced episode to that experienced after handling moldy hay. In all three there was a fall in vital capacity (VC) and forced expiratory volume in one second (FEV,), and a delayed pattern was seen at 12 hr (Table II). One other patient (A. N. J.) produced an immediate response with a fall in FEV, and VC at 30 min, which we consider to be caused by his asthmatic condition. Response to challenge complement-consuming
vs capacity
It can be seen that the response to challenge is not “predicted” by the complement-consuming capacity of the serum for M. fueni antigen (p > 0.1, MannWhitney U test). This was most marked in two patients (Table II): D. R. G. responded to challenge with a complement-consuming capacity of 40 CH50 units/ml of serum, whereas E. G. L., with a complement-consuming capacity of 1630 CH50 units/ ml of serum, failed to respond to challenge. Ten of the 16 patients had values between 40 and 1600 CH50 units/ml of serum, and of these two (E. R. J. and E. P.) reacted with a complement-consuming capacity of 750 and 710 CH50 units/ml, respectively. Interestingly, patient E. P. responded to a very small dose of antigen (700 pg), and considering that not all the nebulized material reached the lungs by virtue of condensation and expiration, it shows that for some highly sensitized individuals an extremely small dose can provoke a reaction. Changes levels
in complement
levels and antibody
There were no significant increases or decreases in CH50 levels over the first 6-hr period after challenge for either responding patients (two tested) or nonresponding patients (12 tested) (Fig. 1). Moreover,
VOLllME 68 NUVBER I
TABLE -_. --
inhalation
II. Response
to
inhalation
challenge
with
M. faeni
purified
challenge,
skin
KH50) 40
2 mg
E. :; J E. I’.
1s 7 IO
2mg 700 pg
I. ‘,I H. 3 E. I. J. A. \.J
‘OS 243 56 IS
1 1 I.2 1.3
w mg mg mg
+ Starting at 6 hr, peak 14 hr Mild at 6-hr. severe 15-hr -
0 Y 85 1630 34
2.3 1.7 2.2 2 2.1
mg mg
-
mg
-
+
++--
-
mg
-
+
++--
-
mg
-
2 mg
-
+
++--
-
+ +
++-+---
+ Starting at 5 hr, lasting
+
+
E. T. C. E. F.
Name 1 c-i.
!“I. J. ‘\. R. 0. -1. J ci. L. i.
11. .‘. J.
54
Ii. 0,
G.
80
L. ,. E. I< J
66 750
2.8 mg 2 mg 2w
Response
test
10 min
2% hr
6 hr
24 hr
36 hr
+
-
-
-
-
-
= negative;
MEFR
= maximum
expiratory
+
+ + +
++-++-++--
i
-
--
-
Fall in spirometry 6i MEFR
+
-
61
- Fall in spirometry (12 hr) -- Immediate asthmatic reaction -. -
-
-----_____
Maximum temperetures (“Cl
Precfpitins against M. faeni
39.5 at i 2 hr
4 +
39.8 at I? hr
+ +-k
hr)
++ 4
t+
*+
c
+ c+ +
None but cough & dyspnea present -
-
- Fall in FEV,, in- 38.3 at X hr crease in obstructive
pattern
t + ii+
---
Row rate.
there was no decline in complement-fixing antibody in two of the responding patients (D. R. G. and E. P.) (Table III). All sera with values of >30 CH50 units/ml produced a positive precipitin response. This is ?Jatistically significant (p < 0.01). Skin test results
Lung function after challenge
(I2
13 hr
-_. + .: positive;
Skin
+
lung
extract
Challenge dose of M. faeni extract
D.
in farmer’s
--. Complement consumption maximum
--.
test
and biopsies
The skin-test results were similar to those experienced previously. fi For the challenged patients, 11 of the 16 produced an immediate skin reaction of central edema, sometimes with pseudopods and surrounding er) thematous zone between 2 and 10 min. The edematcus response was up to 3 by 2 cm in area (patient D R. G .). The skin reaction resolved in one patient at 211: hr, but for eight of the 11 the maximal time of inrvolvement was 2 to 3 hr; thereafter the reaction pelsked at 6 hr. All skin tests were negative or had re!.idual involvement only at 24 and 36 hr. There were only marginal qualitative differences between the reactions to proteins or glycopeptide antigens. For the recently diagnosed patients, whose skin tests were observed only up to 4 hr (excluding two patients on stczroids), six of eight were positive at 15 min, five of se ien were positive at 1 hr, three of four were positive
at 2 hr, and two of three were positive at 4 hr. Three biopsies performed at 10 min revealed edema as the main feature; in only one biopsy was diapedesis of eosinophils seen in several small vessels. An interesting correlation was observed (Table II) between positive immediate skin-test responses and complement-consuming capacities of 40 or more CH50 units/ml of serum (p = 0.0002). DtSCUSSlON
The results show that the complement-consuming ability of farmer’s lung serum does not forecast the capacity of an individual to respond to inhalation challenge using relatively low doses of M. ,faeni antigen with an episode of farmer’s lung. Although this might have been predicted from the presence of healthy farmers with precipitins against M. faeni in a farming population, 26 it was possible that the&seantibodies were of the poorly or non-complement-fixing subgroups of IgG (i.e., IgG2 and IgG4). Our results do not appear to confirm those of de Ridder and Berrens,27 who were able to differentiate between precipitin-positive asymptomatic pigeon breeders and symptomatic cases of pigeon breeder’s
52
J. ALLERGY CLIN. IMMUNOL. JULY 1981
Edwards and Davies
3
2
1
h rs.
after
4
exposure.
1. Complement levels after challenge. Responding no general trend in CH50 levels for either responding
FIG.
TABLE III. Complement consumption per milliliter serum of two responding challenge patients with M. feani antigen at zone of equivalence (1 mg/ml)* Time
after
challenge (hrl
D. R. G.
E. P.
Start 2 4 6 I4 24
40 47.9 40.5 ND 44.4 43.6
710 730 760 775 ND 775
ND = not done. *Note no fall in antibodies during
24 hr after
capable
of complement
consumption
challenge.
lung on the basis of complement-consumption tests. Moore et a1.28 also found generally higher titers of complement-fixing antibodies in symptomatic pigeon breeders. However, the work presented here and our relevant inhalation challenge studies in humidifier fever2s indicate that in these two diseases patients who respond to challenge do not necessarily have higher titers of complement-consuming antibodies than nonresponders. Perhaps pigeon breeder’s lung differs in
5
6
14 Fig. la.
patients are asterisked. Note that there is or non-responding challenge patients.
this respect from farmer’s lung and humidifier fever; indeed, the failure of M. fueni and humidifier fever antigens29 to reduce complement levels in both responders and non-responders differs from the results of Moore et a1.28These authors found that complement levels of precipitin-positive asymptomatic pigeon breeders fell after challenge, whereas the complement levels of symptomatic cases were not affected. We have not evaluated the immunoglobulin(s) responsible for the immediate skin reactions seen in 11 of the 16 patients, but Patterson et a1.30 have shown that IgE antibody against M. faeni was not a feature of the farmer’s lung cases they studied. There is therefore the possibility that the immediate skin reactions were caused by IgG4 involvement.31 What then is the nature of the response to the inhaled material? It is unlikely to be alternative pathway complement activation, since M. fueni extracts prepared in our laboratory did not activate the alternative pathway even at ten-times the concentration used.32 Also, direct stimulation of macrophagesLB is unlikely, since one would expect a more uniform response to the challenge doses if this were so. If complement-fixing antibodies in lung secretions are the same as those in circulation, then correlation might be expected between complement-consuming
VOL. JME 68 NUlilBER 1
capacity and response for immune complex-induced epliodes. Deuschl and Johansson33 found an average of 8% IgG to be produced locally in the lungs of normal individuals, but the percentage varied greatly when groups with known bronchopulmonary disease were tesed; figures for extrinsic allergic alveolitis were not inciuded, and IgM levels were not determined. ‘The time course of the responses was not that of an lgE,-mediated reaction. Although 11 of 16 patients ga*‘e immediate skin test responses, in only one of thei;e was there evidence of a decrease in pulmonary fuI!ction within the first 30 min after challenge. We car postulate a cell-mediated immune response, and Hansen and Penny3” consider cell-mediated immunity to ‘)e a feature of pigeon breeder’s lung, but the skin reactions observed were not of the delayed type. However, skin reactions may not reflect the immunologic capacity and/or responsiveness of the lung, considering the observation of negative skin tests but po!,itive response to nasal challenge with mite extrat:ts.35 The time course of a delayed reaction (18+ hr n skin) may well be shortened in the lung because of the relatively greater numbers of lymphocytes in situ or derived from the circulation. We have shown pu monary responses to purified protein derivative of M:I cobacterium tuberculosis (BCG) in BCG-immunixed rabbits at 3 and 9 hr,36 but this was not the experience of Richerson with aerosol challenge of tht; protein derivative in guinea pigs sensitized with Frt:und’s complete adjuvant (containing killed M. tubcwulosis organisms), where at 6 hr there were only mElima lesions. Perhaps the difference was in the do”;e administered. 4n increasing number of laboratories emphasize the role of the lung as an almost independent organ immunologically. Hunninghake and Fauci38 have shown that 10” sheep red blood cells introduced intrauacheally into guinea pig lungs produced plaqueforming cells (complement-fixing antibody-producing ceils) in the hilar lymph node. At 10 X log sheep red blrlod cells introduced intratracheally, plaque-forming ceils were found in lungs, hilar lymph nodes, and to a IeFser extent the spleen, whereas with 20 X log sheep red blood cells the spleen became the major organ for phque-forming cells of the lungs, hilar lymph node, an 1 spleen, There seems to be a critical dose above which antigenic stimuli spill over into the circulation, anl this may involve active transport of antigens (or bn:akdown products) by macrophages to other lymphoid tissue. For farmer’s lung the inhaled material is mainly particulate, 39 and our work on the respirable fraction of moldy hay shows that the soluble material in respirable moldy hay dust, although capable of activat-
Inhalation
challenge,
skin test in farmer’s
lung
63
ing Cl directly,“” does not contain antigens of M. fueni as judged by the failure of concentrated extracts of the soluble material to react with farmer’s lung serum. The particulate material contains antigens that absorb out antibodies to the cell wall of M. @eni (unpublished observationj. Thus the antigenic components of moldy hay dust, since they are not soluble and diffusable, are trapped in the lungs, and experimentally the macrophage is the initial phagocytic cell.-‘O
There are therefore good reasons for the development of local immunity, certainly in farmer’s lung. Subsequent inhalation challenge would then involve immunoglobulins and cells present within the lung and might not necessarily be reflected in the circulation, particularly IgA and 1gE.I”’ Evidence for local pulmonary immunity has been provided experimentally by Kawai et a6., ii with use of bronchial la.vage cells from rabbits immunized intratracheally with thermophilic actinomycete antigen, and in human disease by Schuyler et ai. .I1 who demonstrated macrophage migration inhibitory factor to be produced by bronchial lavage cells from a patient with pigeon breeder’s lung incubated with pigeon serum or pigeon droppings extract. Peripheral lymphocytes did not produce migration inhibition factor under the same conditions. When challenged with pigeon serum the patient responded with an attack of pigeon breeder’s lung, and afterwards the bronchial lavage cells were refractory for up to 7 days. The refractory penod was noticed by Williams? in his classic study of inhalation challenge in farmer’s lung with
moldy
hay extracts
and may be the result
of
several factors such as persistence of antigen in the lung? The depletion of intracellular biologically active factors could also cause a refractory period, which would fit well with our observations on humidifier fever,4’:’ where pyrexial episodes occurred on the first day back to work after a break; a build-up of factors such as lymphocyte pyrogen Factor-” had occurred. It therefore seems that local immunity m the lung provides many answers to the problems encountered in extrinsic allergic alveolitis, and studies employing bronchiolar lavage technique should provide solutions to some of these outstanding problems We expressour appreciation for the help given by Drs. G. 0. Thomas and A. Axford, Machynlleth Chest Clinic. Dr. G. 0. Thomas, Neville Hall Hospital. Abergavenny. and Dr. D. J. Jones, Withybush Hospital. Ilaverfordwest. REFERENCES
1. Ramazzini B: De morbus artificum. Chicago. 1940. University of Chicago Press.
64
Edwards
and
J. ALLERGY CLIN. IMMUNOL. JULY 1981
Davies
2. Williams JV: Inhalation and skin tests with extracts of hay and fungi in patients with farmer’s lung. Thorax 18:182, 1963. 3. Reed CE, Sosman A, Barbee RA: Pigeon breeders’ lung. JAMA 193:81,, 1965. 4. Riddle HF, Channel1 S, Blyth W, Weir DM, Lloyd M, Amos WMG, Grant IWB: Allergic alveolitis in a maltworker. Thorax 23:271, 1968. 5. Amow PM, Fink JN, Schleuter DP, Barboriak JJ, Mallison G, Said SI, Martin S, Unger GF, Scanlon GT, Kurup VP: Early detection of hypersensitivity pneumonitis in office workers. Am J Med 64:236, 1978. 6. Edwards JH, Baker JT, Davies BH: Precipitin test negative farmer’s lung: activation of the alternative pathway of complement by mouldy hay dusts. Clin Allergy 4:379, 1974. 7. Banaszak EF, Thiede WH, Fink JN: Hypersensitivity pneumonitis due to contamination of an air conditioner. N Engl J Med 283:271, 1970. 8. Pepys J, Riddell RW, Citron KM, Clayton YM: Precipitins against extracts of hay and fungi in the serum of patients with farmer’s lung. Acta Allergol 16:76, 1961. 9. Pepys J, Jenkins PA, Festenstein GN, Gregory PH, Lacey ME, Skinner FA: Farmer’s lung, thermophilic actinomycetes as a source of “farmer’s lung hay” antigen. Lancet 2:607, 1963. 10. Pepys J, Jenkins PA: Precipitin (FLH) test in farmer’s lung. Thorax 2Or21, 196.5. 11. Pepys J: Monographs on allergy. Basel, 1969, S. Karger. 12. Teisberg P, Fauchald P, Flatmark A, Myhm E, Mellbye DJ, Jansen H, Brodwall E: Serial complement studies in a patient with Goodpasture’s Syndrome treated with bilateral nephrectomy and renal transplantation. Am J Med 59563, 1975. 13. Barrowcliff DF, Arblaster PG: Farmer’s lung: a study of an early acute fatal case. Thorax 23:490, 1968. 14. Arthus M: Injections rep&es de serum du cheval chez le lapin. Compt Rend Sot Biol 55:871, 1903. 15. Ghose T, Landrigan P, Killen R, Dill J: Immunopathological studies in patients with farmer’s lung. Clin Allergy 4:119, 1974. 16. Wenzel F, Emanual DA, Gray RL: Immunofluorescent studies in patients with farmer’s lung. J ALLERGY CLIN IMMUNOL 4Br224, 1971. 17. Kawai T, Salvaggio J, Harris JO, Arquembourg P: Alveolar macrophage migration inhibition in animals immunized with thermophilic actinomycete antigen. Clin Exp Immunol 15: 123, 1973. 18. Schorlemmer HU, Edwards JH, Davies P, Allison AC: Macrophage responses to mouldy hay dust, Micropolyspora fueni and zymosan, activators of complement by the alternative pathway. Clin Exp Immunol 27:198, 1977. 19. Gel1 PGM, Coombs RAA, editors: Clinical aspects of immunology. Oxford, 1963, Blackwell Scientific Publications, Ltd. 20. Weissman G, Zurier RB, Spieler PJ, Goldstein IM: Mechanisms of lysosomal enzyme release from leucocytes exposed to immune complexes and other particles. J Exp Med 134: 149S, 1971. 21. Hawkins D: Neutrophilic leukocytes in immunologic reactions: evidence for the selective release of lysosomal constituents. J Immunol 108:310, 1972. 22. Ovary 2, Bier OG: Quantitative studies on passive cutaneous anaphylaxis in the guinea pig and its relationship to the Arthus phenomenon. J Immunol 71:6, 1953. 23. Edwards JH: The double dialysis method for producing farmer’s lung antigens. J Lab Clin Med 79:683, 1972.
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Edwards JH: The isolation of antigens associated with farmer’s lung. Clin Exp lmmunol 11:341, 1972. Kabat EA, Mayer MM: Experimental immunochemistry. Springfield, Ill., 1961, Charles C Thomas, Publisher. Grant IWB, Blyth W, Wardrop VE, Gordon RM, Pearson JCG, Mair A: Prevalence of farmer’s lung in Scotland: a pilot survey. Br Med J 1:530, 1972. de Ridder G, Berrens L: Complement consumption in the diagnosis of pigeon breeder’s disease. Stand J Respir Dis 60:243, 1979. Moore VL, Fink JN, Barboriak JJ, Ruff LL, Schlueter DP: Immunologic events in pigeon breeders’ disease. J ALLERGY CLIN ~IUNOL 53:319, 1974. Edwards JH, Cockcroft A: Inhalation challenge in humidifier fever. Clin Allergy. (In press.) Patterson R, Roberts M, Roberts RC, Emanuel DA, Fink JN: Antibodies of different immunoglobulin classes against antigens causing farmer’s lung. Am Rev Respir Dis 114:315, 1976. Vijay HM, Perelmutter L: Inhibition of reagin-mediated PCA reactions in monkeys and histamine release from human leukocytes by human IgG4 subclass. Int Arch Allergy Appl Immunol 53:78, 1977. Edwards JH: A quantitative study on the activation of the alternative pathway of complement by mouldy hay dust and thermophilic actinomycetes. Clin Allergy 6:19, 1976. Deuschl H, Johansson SGG: Immunoglobulins in tracheobronchial secretion with special reference to IgE. Clin Exp Immunol 16:401, 1974. Hansen PJ, Penny R: Pigeon-breeder’s disease. Study of the cell mediated immune response to pigeon antigens by the lymphocyte culture technique. Int Arch Allergy 47:498, 1974. Huggins KG, Brostoff J: Local production of specific IgE antibodies in allergic rhinitis patients with negative skin tests. Lancet 2: 148, 1975. Edwards JH: An investigation of the antigenic complexes associated with farmer’s lung and allied diseases and their pathogenicity. University of Wales, 1968, Ph.D. Thesis. Richerson HB: Acute experimental hypersensitivity pneumonitis in the guinea pig. J Lab Clin Med 79:745, 1972. Hunninghake GW, Fauci AS: Immunologic reactivity of the lung. V. Regulatory effects of antibody on the pulmonary immune response to locally administered antigen. J Immunol 118:1728, 1977. Edwards JH: The effect of soluble material from mouldy hay dust on the complement system. Immunol 37:91, 1979. Edwards JH. Wagner JC, Seal RME: Pulmonary responses to particulate materials capable of activating the alternative pathway of complement. Clin Allergy 6:155, 1976. Schuyler MR, Thigpen TP, Salvaggio J: Local pulmonary immunity in pigeon breeder’s disease. Ann Int Med 88:355, 1978. Salvaggio J, Phanuphak P, Stanford R, Bite D, Claman H: Experimental production of granulomatous pneumonitis. J ALLERGY CLIN IMMUNOL 56:364, 1975. Edwards JH: Microbial and immunological investigations following an outbreak of humidifier fever and remedial action. Br J Ind Med 57:35, 1980. Chao P, Francis L, Atkins E: The release of endogenous pyrogen from guinea pig leukocytes in vitro. J Exp Med 145:1288, 1977.