Basophil arrival and function in tissue hypersensitivity reactions

THE JOURNAL

OF

ALLERGY AND

CLINICAL VOLUME

IMMUNOLOGY

64

NUMBER 2

Mechanisms interactions

of hypersensitivity:

Basophil arrival hypersensitivity Philip W. Askenase,

and function reactions

Cellular

in tissue

M.D. New Haven, Corm.

Bone murrow-derived blood basophils are recruited into the tissues by immune mec~hanismsin a tariclty of delayed time-course hypersensitivity responses. In the skin these are c~c~fl~d cutantwus basophil hypersensitivity (CBH) reactions. In guinea pigs, it is now established that the elicitation of’ CBH is dtipendent on T cell- andlor B cell (antibody)-triggered mechanisms. Both are subject to modulation. T ceflLmediated CBH seems to be suppressed in hasophil-poor tuberculin-type reactions. B cells mediate CBH via antibody of IgG , isotypc through mec,hanisms that invof\,e Fc receptors, which tnn be competitively blocked. After basophifs urrivr at (I CBH ,-cwc~tionthc,y can be triggered by antigen ta immediutefy refeuse mediators such as histamine. Thus, one consequence of the arrival and accumulation of basophils at delayed hypersensitivilr reuctions is ta augment the anaphyfactic potential qf‘a given tissue site. In reactions to parasites. release oj mediat0r.r by tissue hasophifs seems to aid in the expulsion c?f’these muftic~~flufar organisms. in addition, histamine released bv recruited busophils, or by focafl~ resident mast cells, may modulate some delayed reactions through stimulation of histumine-2 rewptors OIZcells suc,h as T lymphocytes. In mice, mast cell release of serotonin and subsequent stimulation of the lotal vasculaturc~ seems to be required to alloM. diupedesis and tissue acwmufation of vurious f?one marrow-derived accessory leukocytes in delayed-type hypersensitivity responses. Thus, busophils and must cells, und their release c$ rnediutors suc.h as vusouctive amines, are involved in the onset, development, ancfjidnction of’ various tissue hypersensitivit! responses.

Received for publication March 30, 1979. Accepted for publication March 30, 1979. Reprint requests to: Philip W. Askenase, M.D., Department of Medicine, Yale University School of Medicine, 333 Cedar St.. New Haven, CT 06510.

From the Department of Medicine, Yale University School of Medicine. This work was supported in part by grants from the United States Public Health Service (No. AI- 12211 and No. AI- 11077) and the American Cancer Society (No. IM-70E). Presented at the Postgraduate Course in Allergy and Clinical Immunology at the meeting of the American Academy of Allergy in New Orleans, La., March, 1979. 0091-6749179/080079+11$01.1010

@ 1979 The C. V. Mosby

Co.

Vol. 64, No. 2, pp. 79-89

J. ALLERGY

80 Askenase

Abbreviations used: cutaneousbasophil hypersensitivity thymic-derived lymphocytes lymphocytes derived from the mammalian equivalent of the avian bursa of Fabricius PHA phytohemagglutinin Con A concanavalin A IFA incomplete Freund’a adjuvant CFA complete Freund’s adjuvunt KLH keyhole limpet hemocyanin PCA passive cutaneousanaphylaxis DH delayed (tuberculin-type) hypersensitivity PPD purified protein derivative of tuberculin DTH delayed-type hypersensitivity S-HT serotonin VAA vasoactive aminea MAO monoamineoxidase

CBH T cells B cells

Basophils arise in the bone marrow and are normally found in the marrow and blood, but not in the extravascular tissues. In the blood of normal humans and guinea pigs they are the least common of the leukocytes, comprising about 0.5% of the total, or about SO/cu mm. However, it is now well established that basophils can be recruited to enter the tissues from the blood. Recruitment of basophils into the tissues occurs in an immunologically specific manner in a variety of delayed time-course hypersensitivity responses.‘. ” In the skin these are called CBH reactions. Basophil accumulations also occur in immunologic reactions occurring in the gastrointestinal tract,:‘-~Xthe eye,” the kidneys,“‘* ” and probably the nasal mucosa.lZ lmmune recruitment of basophils to extravascular tissue occurs in contact hypersensitivity, ‘+I; allergic rhinitis,” and graft rejection,“* lx and also in responses to proteins,‘“-” erythrocytes,2’1 viruses.“’ fungi,“,’ tumors,‘fi round worms,-‘. ,‘x ‘. ” and insects2x-“0 (Table I). Thus, it is evident that tissue basophil hypersensitivity responses occur in a variety of common and clinically relevant circumstances, and are thought to have significant biological consequences. This review will focus on four aspects of these basophil reactions: (1) how basophils are immunologically recruited to arrive in the tissues; (2) the apparent regulation of tissue basophilia; (3) how basophils are activated to anaphylactic function after arrival in the tissues; and (4) the clinical consequences of basophil arrival and function in delayed reactions. In addition, the role of mast cells in delayed-type hypersensitivity will be considered, and a working hypothesis will be presented whereby basophils

CLIN IMMUNOL. AUGUST 1979

TABLE I. Cutaneous basophil hypersensitivity

(CBH)

Basophils [% of total infiltrate) Type of reaction

Guinea

Jones-Motereactions (sensitization by protein antigen in saline or IFA) Contact hypersensitivity (DNCB, oxazolone, poison ivy, nickel, etc.) Viral hypersensitivity Parasite(schistosome) reactions Insect (tick) reactions Tumor rejection Allograt’t rejection Tuberculin reactions (sensitization with CFA or live mycobacteria)

Human

pig

20-65

5- 10

35-5s

Y-16

74-90 12-n 35 o-25

‘, ‘) s-10 o- IO

DNCB = dinitrochlorobenzene. and/or mast cells might have either positive or negative (suppressive) effects in delayed responses. Immune recruitment CBH reactions

of basophils

to

The induction of CBH, like most immunologic phenomena, appears to depend on T cells. Following immunization of guinea pigs, there is proliferation in the paracortical T cell- dependent regions of draining lymph nodes. ” These nodes contain cells which proliferate in vitro when stimulated with the immunizing antigen.E’2These responses are probably a measure of sensitized T cells. Thus far only “T cell-dependent” antigens have been shown to induce CBH. When synthetic polyaminoacid polymer “proteins” are used for immunization of responder vs nonresponder guinea pigs, CBH is induced only in animals with responding T cells .x:S It is thus clear that the induction of CBH is T cell dependent. However, the elicitation of CBH has been shown to be dependent on T cell nr B cell mechanisms (Fig. 1). The role of T cells in the elicitation of CBH is clear from the following: (1) CBH-like reactions result from intradermal challenge of guinea pigs with T cell mitogens (PHA, Con A) and plot with B cell mitogens (pokeweed, lipopolysaccharide.“(2) The elicitation of CBH in guinea pigs sensitizec and tested with ovalbumin is abolished by treatmen with an antithymocyte serum, as are cutaneous base phi1 responses to PHA and Con A.“” (3) Systemi

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64 2

Basal cells

REGULATION OF TISSUE BASOPHILIA

TISSUE BASOPHIL ARRIVAL

81

TISSUE BASOPHIL FUNCTION

CFA SUPPRESSION *----PROTECTION ,? PARASITES,

T CELLS EASOPHIL ACCUMULATION B CELLS-7S-I-

-

CUTANEOUS BASOPHIL ANAPHYLAXIS

/ \

IgG, c-----

REGULATION 17 “IA HlDTAMlNE RECEPTORS1

Fc COMPETITION

FIG. 1. An overview of cutaneous basophil reactions. Blood basophil accumulation in the tissues is guided via T cell or antibody (IgG,) mechanisms. T cell CBH is regulated through suppressive factors generated by CFA immunization, and antibody CBH can be blocked via competition for host Fc receptors. After arrival, basophils can be triggered to release mediators like histamine, which can serve protective or regulatory functions.

passive transfer of conjugate-specific CBH reactions occurs with nylon nonadherent lymphocytes containing 97% to 98% surface immunoglobulin-negative cellsZj6, and local passive transfer is achieved with sensitized T cells of similar purity.a7 In addition to T cell-mediated CBH elicited by hapten-carrier conjugates or by carrier proteins, CBH reactions can be elicited in an entirely hapten-specific manner. These delayed time-course, basophil-containing reactions feature erythema and thickening in the skin and are transferable by immune serum antibodies, and thus are examples of B cell-dependent CBH (Fig. 1). Remarkably small doses of serum are able to mediate these reactions. With adult guinea pig recipients and systemic transfers, the limiting transfer dose is about 0.25 ml of immune serum; with neonatal recipients the limiting transfer dose is 0.03 m1.3X*39In addition, small numbers of highly purified immune guinea pig B cells (3 x lo6 cells, 98% pure) can mediate CBH reactions when mixed with antigen and used for local passive transfers to inbred guinea pigs.:j7 These B cell transfers of CBH may be caused by local secretion of minute amounts of antibody. A variety of immunization procedures with several antigens has provided serum which transfers CBH. Donors can be immunized by contact sensitization or with proteins emulsified with IFA or CFA, or with no adjuvant at all. The oxazolone and picryl (trinitrophenol) determinants can readily induce and elicit these reactions, as can the native protein keyhole limpet hemocyanin (KLH).“. 3x-*o Although it is established that these reactions are mediated by serum antibodies, knowledge of the factors which lead to the generation of these unusual antibodies is incomplete. Thus far it has not been possible to generate dinitrophenyl (DNP)- specific antibodies which can mediate CBH, in contrast to success with trinitrophenyl

(TNP). The facility of inducing B cell CBH to some determinants and not to others is an interesting question that is currently under study. Differences in immunodominance or differences between immunization regimens could be an explanation and might also account for the early failures of others to find B cell CBH reactions. However, the ability of antibody to mediate CBH has now been confirmed by Askenase and Dvorak.‘” Chromatography analysis using Sephadex G200 and DEAE cellulose has revealed that hapten-specific CBH reactions are mediated by factors occurring in 7SIgGi fractions of immune serum.3g There are two principal IgG antibody subclasses in guinea pigs. IgG, antibodies are complement fixing and mediate Arthus-like reactions (type III hypersensitivity).4’ IgG, antibodies passively sensitize skin mast cells and thus mediate passive cutaneous anaphylaxis (PCA) reactions (type I hypersensitivity).-‘” The serum factors which mediate CBH can be removed by a hapten-coupled immunoadsorbent column and subsequently eluded with monovalent hapten. As little as 30 pg purified antibody obtained in this manner is sufficient to passively transfer systemic sensitization to neonatal guinea pigs (weight. 100 gm). When purified antibody is run through immunoadsorbent columns coupled with anti-IgGi vs anti-lgGz sera of high specificity, the anti-IgG, and not the anti-IgGz column removes the activity. XI Thus, by all conventional criteria, serum factors mediating hapten-specific CBH reactions appear to be 7SIgG, antibodies (Fig. 1). An unusual and as yet unexplained property of antibodies which mediate CBH is their relatively low affinity. Suitable antiserum can be obtained 1 wk after immunization, when low affinity antibodies predominate. Boosting procedures which raise antibody titers and overall affinity fail to substantially increase the

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CLIN. IMMUNOL. AUGUST 1979

FIG. 2A. When basophils arrive at a CBH reaction 24 to 48 hr after intradermal challenge with a soluble protein test antigen (KLH), the cells demonstrate a smooth unactivated surface and are not degranulated. These basophils which have accumulated in the tissues resemble normal blood basophils. (x 13,800.)

strength of limiting volumes of serum to transfer these reactions. Affinity determinations performed using purified antibody in direct binding equilibrium dialysis have revealed that the average affinity constant of antibodies mediating CBH is 8 x 104M-‘.a9 This is a rather low affinity and contrasts with antibodies mediating PCA or complement fixation which generally have 100 times greater affinity.“, ‘5 These immunochemical findings on antibodies mediating CBH have presented two new puzzles. Firstly, how do antibodies of such minimal affinity mediate a rather substantial biologic reaction which features a delayed time course of macroscopic and microscopic components? In addition to the basophil infiltrates by which these antibody-mediated reactions are usually characterized, approximately 10% to 45% of the infiltrating cells are eosinophils.’ The remainder are mononuclear cells. Many of these appear to be lymphocytes and some are transformed lymphoblasts.” In addition, deposits of fibrin (which are unusual in cell-mediated CBH reactions in guinea pigs”“) have been noted in 1-p plastic embedded sections and in frozen sections stained with fluoresceinconjugated antifibrin antibody.”

In summary, T cells clearly mediate CBH. In addition, small quantities of 7SIgG, anaphylactic-type antibody of low affinity also mediate CBH, in that they generate a delayed time-course inflammatory reaction consisting of several diverse components, including basophil infiltrates.

Regulation

of tissue

basophilia

The accumulation of basophils in delayed reactions appears to be under complex regulation. Both T cell and antibody mechanisms can guide the arrival of basophils, and both are subject to modulation (Fig. 1). Suppression of T cell-dependent CBH is best exemplified in tuberculin-type reactions of guinea pigs immunized with CFA. Basophils are exceedingly rare in delayed responses of these animals, which are thus called DH, in contrast to CBH responses of animals immunized with IFA. However, sensitized lymphoid cells or enriched T cells from animals immunized with CFA can transfer to normal recipients the ability to produce antigen-specific, basophil-rich, delayed responses that were no1 obtained in the donors.:‘” Moreover, cultured lymphoid cells from animals immunized with CFA and mani-

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Basal cells

83

FIG. ZB. When a small amount of specific antigen (2 pg KLH) is reinjected into such a 24-or 48-hr CBH reaction the basophils are triggered to function immediately (within 60 set). These cells are stretched out and demonstrate an activated surface consisting of long and tortuous filopodial projections. In addition, there is obvious degranulation with extrustion of granules by exocytosis, and anaphylactic release of histamine. (x9,800.)

festing basophil-poor DH responses appear to elaborate basophil chemotactic lymphokines in quantities similar to cells from donors immunized with IFA and manifesting CBH. -(’ Thus, T cells which are able to recruit basophils to the tissues are present but inoperative in animals immunized with CFA, or the basophil chemotactic lymphokines they elaborate are inoperative. This indicates that T cell CBH is suppressed (Fig. 1). The mechanism of this suppression is unknown, but it is antigen-specific and localized to the site of skin testing, as animals can be sequentially immunized for DH and CBH to different antigens and can then manifest each reaction at separate flank sites.“8 Possible examples of a luck of suppression of T cell CBH include: (1) CBH occurring early after CFA immunization, then evolving into basophiltuberculin-type DH reactions via supprespoor, sion “. “, 4e-sn;(2) CBH in neonates following either IFA or CFA immunization”“; (3) profound CBH-like responses elicited in neonates by PHA skin testing, then evolving after 10 to 14 days into moderate CBH, perhaps via suppression”3; and (4) basophil responses equivalent to human Jones-Mote- type responses in some, but not all, human PPD reactions.2’

Antibody-mediated CBH is also subject to regulatory factors. The most clearly identified thus far involve competition for Fc receptors via which these antibodies mediate CBH (Fig. 1). IgG, antibodies, which mediate CBH in guinea pigs, also mediate PCA reactions via antigen-independent cytophilic attachment to Fc receptors on cutaneous mast cells. Thus, treatment of animals with immunoglobulins containing Fc portions able to interact with such receptors could interfere with antibody-mediated CBH reactions. Since rabbit IgG antibodies mediate PCA reactions in guinea pigs by the ability of the Fc portion of these immunoglobulins to interact with cutaneous mast cells,” -I Fc competition experiments were constructed. Administration of rabbit IgG- inhibited antibody transferred CBH in a dose-response fashion. Human (but not bovine or sheep) gamma globulin also had the ability to inhibit antibody-mediated CBH. Rabbit and human (but not bovine or sheep) gamma globulin also inhibited PCA reactions, as previously noted.“” Subsequent studies employing enzymatic digestion of rabbit IgG showed that purified Fc fragment alone could compete with the ability to transfer antibody-mediated CBH, while the corre-

84

J. ALLERGY

Askenase

HISTAMINE , __ RECEPTORS

-2 s -.

,’

‘\

/’ DTHY SUPPRESSOR T CELL (Ly 23+) I

\

&L&p NEGATIVE SUPPRESSOR LOOP

DTH INDUCER T CELL (Ly I+)

\ \

l POSITIVE EFFECTOR LOOP

1 CHEMOTACTIC AND MIGRATION INHIBITING LYMPHOKINES

J

DTH/

ENDOTHELIUM /

DlAPEDEStS AND TISSUE ACCUMULATION OF BONE MARROW DERIVED AUXILLARY EFFECTOR CELLS

FIG. 3. The hypothesized vasoactive amine loop in DTH. Inducer T cell (Ly 1’) interaction with antigen and consequent release of chemotactic and migration inhibiting lymphokines is viewed as necessary but not sufficient for DTH in mice. In addition, to form a positive effector loop, T cells interact with mast cells which release 5-HT which causes endothelial cells to allow the diapedesis of leukocytes in response to the lymphokines. A negative suppressor loop is formed by the possibility that mast cells also release histamine which acts on histamine-2 receptors of suppressor T cells of the Ly 2+3+ phenotype.

sponding (Fab’), portion of the molecule was inactive.“” PCA tests in animals demonstrating this FC competition showed that the surface of cutaneous mast cells was blocked, but the cells could be discharged by sheep antirabbit gamma globulin. We have concluded from these studies that the Fe portion of antibodies mediating CBH are crucial to their biologic activity and that an Fc receptor on cells in the host is necessary to elicit antibody-mediated CBH reactions. (Fig. 1) The regulation of antibody-mediated CBH via Fc competition has led to a consideration of the relationship between antibody CBH and antibody-mediated cutaneous anaphylactic reactions. In guinea pigs, both antibody CBH and PCA reactions are mediated by IgG, antibodies. An Fc receptor of a host skin cell is also involved in both reactions. The fact that rabbit and human but not sheep or bovine immunoglobulins apparently contain Fc portions which competitively inhibit borh reactions strongly suggests that cutaneous mast cells participate in both processes. However, a PCA reaction is elicitable by local passive transfer and is predominantly an immediate wheal-and-flare reaction which is followed several hours later by an infiltrate of neutrophils and eosinophils,““. STbut not

CLIN. IMMUNOL AUGUST 1979

basophils. ’ In guinea pigs no late macroscopic sequelae are evident following PCA reactions. In contrast, antibody-mediated CBH reactions are not transferrable by local passive means. They require systemic transfers and are characterized by various late components such as macroscopic erythema and thickened skin which contains infiltrates of basophils. eosinophils, and mononuclear cells. In some respects antibody-mediated CBH reactions seem akin to late cutaneous IgE-mediated reactions which are found in ,,umanS..%L59 Late IgE reactions consist of erythema and edema 8 to 24 hrs after a wheal-and-flare reaction elicited by pollen antigens in actively sensitized individuals. They are transferable locally (PrausnitzKiistner transfers), and myeloma IgE is inhibitory via apparent Fc competition.“x Histologically, Iate reactions contain mononuclear cells, eosinophils. basophils, and fibrin deposits.“X, ;19 From the experiments performed to date, a working hypothesis has been constructed for the mechanisms underlying antibody-mediated CBH reactions. It is thought that systemically transferred antibody enters the tissues of the skin and passively sensitizes mast cells via Fc receptors. Following local antigen challenge, a reaction is initiated which may have an initial anaphylactic character, but perhaps due to the low affinity of the participating antibody leads to consequent events which differ from conventional immediate reactions. Perhaps the mast cells elaborate factors which are chemotactic for some or all of the cells found in the reactions, and one of these cells, perhaps the basophil, arrives armed with the transferring antibody similarly adsorbed through its Fc receptor. Upon arrival these basophils meet remaining skin-test antigen and discharge in a manner perhaps similar to the tissue mast cell, thereby releasing more mediators which might further attract the cellular and skin thickening elements of the reactions. In summary, antibody-mediated CBH may be an anaphylactic cascade reaction. However, it has not been ruled out that antibody-mediated CBH may also involve passive sensitization via Fc receptors of other host cells, such as lymphocytes. Anaphylactic function at CBH reactions

of basophils

Once basophils arrive in the tissues can they function to release mediators? This has been studied by challenging 24-hr CBH reactions with additional small amounts of antigen (1% to 2% of the dose of antigen used 24 hr previously to elicit the delayed reaction). Immediate (15 min) consequences of this antigen challenge of a 24-hr CBH reaction are: (1) immediate augmented vascular permeability of an

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Basal cells

64 2

85

SEROTONIN (5HT) RELEASE FROM MAST CELLS IN MURINE DELAYED-TYPE HYPERSENSITIVITY 24 hr DELAYED-TYPE TREATMENT

NONE

HYPERSENSITIVITY

(% positwe

control)

EFFECl POSITIVE CONTROL

RESERPINE

RESERPINE + MONOAMINE OXIDASE INHIBITOR

LOCAL SEROTONIN

GRANULE DEPLETION SEROTONIN + PREVENTION OF 5 HT CATABOLISM TACHYPHYLAXIS OF ENDOTHELIAL SEROTONIN RECEPTORS

COMPETITIVE

CELL

ANTAGONISM

FIG. 4. Three independent

pharmacologic treatments which indicate that mast cell release of 5-HT is required in DTH. Responses are inhibited if mast cells are intracellularly depleted of 5-HT by treatment with reserpine (line21 but the inhibition is reversed if catabolism of released 5-HT is prevented by MAO inhibitor /line 3). Local pretreatment with 5-HT specifically desensitizes the local endothelium to a later 5-HT response and depresses elicitation of DTH (line 4). Finally, a 5-HT antagonist (cyproheptadine) inhibits DTH (line 5). The three manipulations of 5-HT are shown in actively sensitized animals, but similar results are obtained in recipients of transferred immune T cells.

anaphylactic type; (2) significant immediate falls in local tissue histamine; and (3) a 50% decline in the number of identifiable basophils.60 The vascular permeability increases are inhibitable by antihistamines (HI receptor antagonists). Electron microscopy of CBH sites following immediate challenge has revealed the pathognomonic findings of anaphylactic degranulation of basophils (Fig. 2). We have coined the term “cutaneous basophil anaphylaxis” for this phenomenon (Figs. 1 and 2). It is cutaneous because it occurs in the skin; it is dependent on basophils; and it is anaphylactic in character. Thus, one consequence of the arrival and accumulation of basophils at delayed hypersensitivity reactions is to augment the anaphylactic potential of a given tissue site. It is noteworthy that 24 and 48 hr after skin testing with a single injection of antigen, the majority of basophils which arrive have normal-appearing granules (Fig. 2A). However, if additional antigen is locally available, basophils which have arrived can be activated to degranulate by exocytosis (Fig. 2B) and release various mediators including histamine. Antibody appears to mediate this function of basophils at CBH reactions.6’ These findings link the participation of basophils in

delayed reactions with anaphylactic mediator release of immediate hypersensitivity. “Arming” of local tissues for augmented anaphylactic function by basophils probably acts primarily to influence the local tissue environment. The effects of basophil mediators in the tissues could serve a protective function or could regulate immune inflammation (Fig. I). Protection, such as the expulsion of parasites or resistance to tumors, could occur through the release of mediators which can chemoattract effector cells, activate platelets, increase vascular permeability for antibodies and complement, contract smooth muscle, and augment diapedesis of blood-borne effector leukocytes. 62 Alternatively, released histamine could have an immunoregulatory role, particularly through histamine-2 receptors on suppressor T cellsf’“-fi6 (Figs. 1 and 3). The clinical consequences of basophil accumulation at CBH reactions Accumulations of basophils in the skin and gastrointestinal tract is a prominent component of immune responses to multicellular parasites. ’ When guinea pigs sensitized to schistosomes are subsequently rechallenged with infecting schistosome cercariae, the

86

Askenase

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J. ALLERGY

HOURS

40

HOURS

AFTER

BEFORE o----o

CIMETIDINE

BEFORE X---X

AFTER

CONTROL

FIG. 5. Augmentation of delayed hypersensitivity in patients treated with cimetidine, a histamine-2 receptor antagonist. In a double-blind study, patients with acid-peptic disease were treated with cimetidine (300 mg four times daily) or antacids (controls) for 6 wk, and were skin tested with various “recall antigens” at the outset and conclusion of treatment. Induration (in millimeters) at 24 and 48 hr after testing with streptokinase-streptodornase (SKSD) is shown. Augmentation in the cimetidine group vs the controls was significant at 24 and 48 hr (p < 0.005).

site of skin penetration develops punctate erythematous reactions which contain infiltrates of degranulating basophils and edema of the surrounding tissues.2i It is hypothesized that in the initial phase of such reactions, parasite surface coat antigens interact with host T cells or antibody, leading to attraction of various inflammatory cells. Subsequently, antigens shed by the parasites might activate cells that are attracted to the site. Antigen-triggered activation of recruited cells, after they arrive, might also occur via T or B cell mechanisms. In the schistosome reactions it is not yet clear whether basophil recruitment represents a hypersensitivity phenomenon or is associated with protective resistance to the parasites. However, in another system, cutaneous responses to ectoparasites (in particular, ticks), CBH reactions are associated with a profound resistance to the invading organisms .29*‘loV671.6X Guinea pigs are sensitized by allowing 200 tick larvae to feed on the skin. These small, six-footed larvae are about 0.5 mm in length. When released on the skin of guinea pigs they burrow between the hairs and attach to the epidermis via a secreted proteinaceous cement substance. Over the ensuing 2 to 4 days, the mouth parts of the tick are inserted into the uppermost aspects of the dermis. The tick takes a blood meal and then detaches, having increased in size 4- to 7-fold (engorgement). This tick feeding provides a potent sensitization of guinea pigs, such that when another batch of tick larvae are subsequently applied to the

CLIN. IMMUNOL. AUGUST 1979

contralateral flank skin, 90% to 100% are rejected by an apparently immune reaction. This potent, cutaneous immune expulsion of large, multicellular parasites is accompanied by substantial delayed basophil infiltrates around the mouth parts and cement substance of the ticks. A single prior infestation with as few as 10 tick larvae is sufficient to sensitize for this reaction. When the sensitizing dose of feeding tick larvae is greater than 40, immune resistance can last for nearly a year.‘” This CBH-associated immune resistance to ticks can be successfully transferred with immune cells or with immune serum,2”* 30, 6i and often the recipients have resistance that is comparable to that of actively sensitized animals. In fact, as in previous studies employing antibody-mediated CBH to haptens, surprisingly small doses of immune serum (0.5 ml) suffice for passive transfer of virtually complete resistance. How basophil arrival and activation might lead to rejection of the parasites is not known. One possibility is that the ticks commit their metabolic resources to feeding and become fixed to the skin via their cement substance. Then basophils arrive and release various mediators which alter the tissue environment to prevent feeding, leading to death of the ticks from starvation or dessication. In these studies of CBH responses to ticks, it seems likely that T or B cell mechanisms guide the arrival of basophils and perhaps the local functional activation that may participate in expulsion of the organisms. The role of mast cells in delayed-type hypersensitivity

(DTH)

Basophils are one of a variety of bone marrow-derived auxillary effector cells which are recruited from the blood to the tissues in DTH reactions. The finding that blood basophils are recruited to arrive and function in DTH reactions has led to consideration of the role of tissue mast cells in these responses. In contrast to basophils, mast cells are not marrow-derived cells and are not present in the blood, but found normally in the extravascular tissues. Mice appear to lack basophils, but have abundant mast cells in areas of the skin where DTH responses are most easily elicited (i.e., footpads and ears). 69Treatment of mice with a single systemic dose of reserpine prior to challenge significantly suppresses DTH elicited in these sites (Fig. 4).“” Although mast cells in mice contain both histamine and 5-HT, the vessels of mice are insensitive to histamine but quite reactive to 5-HT.” Reserpine depletes mast cells of serotonin, but not of histamine. This suggests that release of the vasoactive amine serotonin by mast cells is required in DTH. The fact that DTH is inhibited in immune cell- transfer recip-

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ients treated with reset-pine suggests that serotonin release by mast cells is regulated by T cells in DTH. Studies with MAO inhibitors suggest that T cells use mast cells in a way that is distinct from the function of mast cells in immediate hypersensitivity reactions. These latter responses are mediated by mast cells and anaphylactic antibodies which are products of B lymphocytes. Reserpine is thought to act by displacing monoamines (such as 5-HT) from binding sites in cytoplasmic granules. Monoamines released intracellularly are then degraded by MAO, but in the presence of MAO inhibitors this catabolism of released monoamines is blocked. Mice treated with reserpine and MAO inhibitors have restored DTH responses (Fig. 4), but the block in immediate-type reactions is maintained.“” Since it is known that the participation of mast cells in immediate hypersensitivity is via a secretory exocytosis of granules which contain mediators,” this suggests that vasoactive mediators in a nongranule cytoplasmic compartment (perhaps in the cytosol) are available for use by T cells. These findings have led to the hypothesis (see Fig. 3) that the early phases of delayed-type reactions are accompanied by a critical T cell to mast cell message whereby 5-HT in the cytosol is released and acts on endothelial cells to open post capillary venules, allowing the transit of marrow-derived precursors into the tissue reactions. Thus, diapedesis in DTH may be amine dependent. These assertions depend on the use of reserpine to deplete 5-HT. Subsequent studies employed two additional and independent pharmacologic manipulations of S-HT. First, 5-HT tachyphylaxis was employed at a DTH reaction to desensitize the vasculature to endogenous release of 5-HT.iU Second, studies employed cyproheptadine, a 5-HT antagonist, which competitively inhibits the interaction of 5-HT with its endothelial receptors.i2 These three independent pharmacologic maneuvers (mast cell 5-HT depletion by reserpine, 5-HT tachyphylaxis of endothelium, and 5-HT antagonism) all decrease DTH in actively sensitized mice (Fig. 4) or in recipients of sensitized T cells, and thus confirm the validity of this hypothesis. The relevance of these findings to other species remains to be determined. In humans and guinea pigs serotonin is not a potent vasoactive amine. However, in DTH reactions of these species there is degranulation of mast cells and endothelial cell activation and gaps,‘” which may be due to vasoactive factors or amines. There is also histamine tachyphylaxis of the endothelium,60 which may indicate endogenous stimulation with histamine. Thus, processes akin to the serotonin-dependent diapedesis identified in DTH of mice may occur in other species.

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Studies on the role of VAA in DTH have led to the formulation of a vasoactive amine loop in DTH (Fig. 3). In this scheme it is proposed that T cells induce DTH reactions. Such inducer T cell(s) in mice express the Ly 1+23- phenotype.” These recirculating T cells normally leave the blood stream and enter the tissues by a process of emperipoliesis (passage directly through the cytoplasm of endothelial cells).‘” When antigen is present in the tissue, rare, passing, specifically sensitized, recirculating T cells become activated, release macromolecular mediators (lymphokines such as migration inhibitory factor [MIF] and chemotactic factors) which lead to the recruitment of various intravascular accessory cells, which are mainly bone marrow-derived and do not ordinarily enter the tissues. It is known that such cells comprise more than 95% of the infiltrate in DTH reactions. x ii However, T cell activation and chemoattractant lymphokine release seem to be necessary. but not sufficient, for the development of DTH. In addition, there is a requirement for T cells to interact with local mast cells, which are normally present in the tissues around small blood vessels. Thus, T cell-mast cell interaction leads to the release of VAA, such as 5-HT. Released amine alters local endothelial cells permitting various intravascular inflammatory cells to enter the tissues in response to the T cell-released chemoattractants. In Fig. 3 these positive influences which amine release might have in DTH are indicated in the bottom half of the diagram. The top half of Fig. 3 shows how a feedback loop is formed by potentially negative or suppressive influences which VAA might have in DTH. Not shown is 5-HT tachyphylaxis, the ability of locally released serotonin to deactivate the endothelium to subsequent doses of 5-HT. This can suppress macroscopic DTH and inhibit the tissue accumulation of leukocytes.“’ The top half of Fig. 3 does depict potential suppression via histamine. Mouse mast cell granules contain both 5-HT and histamine, but the local vasculature is insensitive to histamine. However, T cells in mice have histamine-2 receptors and a variety of in vitro observations suggest that histamine, acting through histamine-2 receptors, may be a mediator of T cell suppression. 63-66 Thus, histamine release by mast cells in DTH of mice, or by basophils and/or mast cells in other species, may act to dampen the reactions via stimulation of histamine-2 receptors on regulatory T cells. In support of this notion, there are clinical observations that DTH responses (PPD, streptokinase-streptodornase [SK-SD], etc.) are augmented in patients with peptic ulcer disease who are treated with cimetidine, a histamine-2 receptor antagonist (Fig. 5 .)7H

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REFERENCES I. Askenase PW: Role of basophils, mast cells and vasoamines in hypersensitivity reactions with a delayed time course. Prog Allergy 23: 199, 1977. 2. Dvorak HF: Cutaneous basophil hypersensitivity. J ALLERGY CLIN IMMUNOL S&229, 1976. 3. Askenase PW. Boone WT, Binder HJ: Colonic basophil hypersensitivity. J lmmunol 120: 198, 1978. 3. Rothwell TLW: Studies of the response of basophil and eosinophil leukocytes and mast cells to the nematode Trichos~t~~n~y/uscohrhr~formis. I. Observations during the expulsion of first and second infections by guinea pigs. J Pathol 116: 15, 1975. 5. Rothwell TLW, Dineen JK: Cellular reactions in guinea pigs following primary and challenge infection with Trichostrongylirs co/ubr@mis with special reference to the roles played by eosinophils and basophils in rejection of the parasite. Immunology 22:733, 1972. 6. Miller HRP: Immune reactions in mucous membranes. II. The differentiation of intestinal mast cells during helminth expulsion in the rat. Lab Invest 24339, 1971. 7. Ogilvie BM, Hesketh PM, Rose ME: Nipposrrong&s brasiliensi.s: Peripheral blood leukocyte response of rats, with special reference to basophils. Exp Parasitol 46:20, 1978. 8. Lee G, Askenase PW, Ogilvie BM: Unpublished observations. 9. Friedlander MH, Dvorak HF: Morphology of delayed-type hypersensitivity reactions in the guinea pig cornea. J Immunol 118:1558. 1977. IO. Colvin RB. Burton JR, Hyslop NE Jr: Penicillin associated interstitial nephritis. Ann Intern Med 81:404, 1974. 11. Calvin RB, Dvorak HF: Basophils and mast cells in renal allograft rejection. Lancet 1:212, 1974. 12. Hastie R, Heroy JH, Levy DA: Basophil leukocytes and mast cells in human nasal secretions and scrapings, studied by light microscopy. (Submitted for publication, 1979.) 13. Dvorak HF, Mihm MC Jr: Basophil leukocytes in allergic contact dermatitis. J Exp Med 135:235, 1972. 14. Dvorak HF, Simpson BA, Bast RC, Leskowitz S: Cutaneous basophil hypersensitivity. Ill. Participation of the basophil in hypersensitivity to antigen-antibody complexes, delayed hypersensitivity and contact allergy. Passive transfer. J Immunol 107:13x, 1971. 15. Wolf-Jurgensen P: Basophilic leukocytes in delayed hypersensitivity. Copenhagen, 1966, Munksgaard. 16. Dvorak HF, Mihm MC Jr, Dvorak AM, Johnson RA, Manseau EJ, Morgan E, Colvin RB: Morphology of delayed-type hypersensitivity reactions in man. I. Quantitative description of the inflammatory response. Lab Invest 31: I1 I, 1974. 17. Askenase PW: Cutaneous basophil hypersensitivity in contact sensitized guinea pigs. I. Transfer with immune serum. J Exp Med 138: 1144, 1973. 18. Dvorak HF: Role of the basophilic leukocyte in allograft rejection. J lmmunol 106:279, 1971. 19. Richerson HB, Dvorak HF, Leskowitz S: Cutaneous basophil hypersensitivity. 1. A new look at the Jones-Mote reaction. J Exp Med 132:546, 1970. 20. Dvorak HF. Dvorak AM, Simpson BA, Richerson HB, Leskowitz S. Karnovsky MJ: Cutaneous basophil hypersensitivity. II. A light and electron microscopic description. J Exp Med 132:558. 1970. 2 I. Richerson HB: Cutaneous basophil (Jones-Mote) hypersensitivity after “tolerogenic” doses of intravenous ovalbumin in the guinea pig. J Exp Med 134630, 1971.

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22. Askenase PW, Atwood EJ: Basophils in tuberculin and “Jones-Mote” delayed reactions of humans. J Clm Invest 58: 1145, 1976. 23. Stadecker MJ, Leskowitr S: The cutaneous basophil response to particulate antigens. Proc Sot Exp Biol Med 142: 150, 1973. 24. Dvorak HF, Hirsch MS: Role of basophilic leukocytes in cellular immunity to vaccinia virus infection. J lmmunol 107: 1576, 1971. 25. Sohnle PC, Kirkpatrick CH: Study of possible mechanisms of basophil accumulation in experimental cutaneous candidiasis in guinea pigs. J ALLERGY CLIN IMMUNOL 59: 171, 1977. 26. Dvorak HF, Dvorak AM, Churchill WH: Immunologic rejection of diethylnitrosamine-induced hepatomas in strain 2 guinea pigs. Participation of basophilic leukocytes and macrophage aggregates. J Exp Med 137:751, 1973. 27. Askenase PW, Hayden BJ, Higashi Cl: Cutaneous basophil hypersensitivity and inhibited macrophage migration in guinea pigs with schistsomiasis. Clin Exp Immunol 23:3 18. 1976. 28. Allen JR: Tick resistance. Basophils in skin reactions of resistant guinea pigs. lnt J Parasitol 3: 195, 1973. 29. Bagnall BG: Cutaneous immunity to the tick hdes hdoqclus. Thesis, University of Sydney, 1975. 30. Askenase PW, Worms MJ: Immune cutaneous basophil resistance to parasite ticks. Fed Proc 38: 1221. 1979. 3 1. Dvorak AM, Bast RC Jr, Dvorak HF: Morphologic changes in draining lymph nodes and in lymphocyte cultures after sensitization with complete or incomplete Freund’s adjuvant. Correlation with immunologic events irr i,ir,o and in culture. J Immunol 107:422, I97 1. 32. Bast RC Jr, Simpson BA, Dvorak HF: Heterogeneity of the cellular immune response. II. The role of adjuvant. Lymphocyte stimulation in cutaneous basophil hypersensitivity. J Exp Med 133:202, 1971. 33. Stadecker MJ, Leskowitz S: Genetic control of cutaneous basophil hypersensitivity. J Exp Med 143:206, 1976. 34. Stadecker MJ, Leskowitz S: The cutaneous basophil response to mitogens. J Immunol 113:496, 1974. 35. Stadecker MJ, Leskowitz S: The inhibition of basophil-rich delayed skin reaction by a heterologous anti-guinea pig T cell serum. J Immunol 116~1646, 1976. 36. Askenase PW: Cutaneous basophil hypersensitivity uncovered in the cell transfer of classical tuberculin hypersensitivity. J Immunol 117:741, 1976. 37. Stashenko PP, Bhan AK, Schlossman SF, McClusky RT: Local transfer of delayed hypersensitivity and cutaneous basophil hypersensitivity. J Immunol 119: 1987, 1977. 38. Askenase PW, Haynes JD, Hayden BJ: Antibody mediated basophil accumulations in cutaneous hypersensitivity reactions of guinea pigs. J Immunol 117: 1722, 1976. 39. Haynes JD, Rosenstein RW, Askenase PW: A newly described activity of guinea pig IgG, antibodies: Transfer of cutaneous basophil reactions. J Immunol 120:886, 1978. 40. Askenase PW, Haynes JD, Tauben D, DeBernardo R: Specific basophil hypersensitivity induced by skin testing and transferred using immune serum. Nature 2%X52, 1975. 41. Askenase PW, Dvorak HF: Unpublished observations. 42. Benacenaf B, Ovary Z, Block KJ, Franklin EC: Properties of guinea pig 7S antibodies. I. Electrophoretic separation of two types of guinea pig 7S antibodies. J Exp Med 117:937, 1963. 43. Ovary Z, Benacerraf B, Block KJ: Properties of guinea pig 7S antibodies. 11. Identification of antibodies involved in passive cutaneous and systemic anaphylaxis. J Exp Med 117:951. 1963.

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44. Siskind GW, Eisen HN: Effects of variation in antibody hapten association constant upon the biologic activity of the antibody. J lmmunol 95:436, 1965. 45. Hurlimann J, Ovary Z: Relationship between affinity of antidinitrophenyl antibodies and their biologic activities. J lmmunol 95:765, 1965. 46. Calvin RB, Dvorak HF: Role of the clotting system in cell mediated hypersensitivity. Il. Kinetics of fibrinogen/fibrin accumulation and vascular permeability changes in tuberculin and cutaneous basophil hypersensitivity reactions. J Immunol 114:377. 1975. 47. Ward PA, Dvorak HF, Cohen S, Yoshida T, Data R, Selvaggio SS: Chemotaxis of basophils by lymphocyte-dependent and lymphocyte-independent mechanisms. J lmmunol 114: 1523, 1975. 48. Leonard EJ. Lett-Brown M, Askenase PW: Simultaneous generation of tuberculin type and basophilic hypersensitivity at separate sites in the guinea pig. lnt Arch Allergy Appl lmmunol. (In press.) 49. Katz Sl, Parker D, Turk JL: B cell suppression of delayed hypersensitivity reactions. Nature 251:550, 1974. 50. Turk JL, Parker D: Further studies on B lymphocyte suppression in delayed hypersensitivity. Immunology 24:75 1, 1973. 51. Neta R. Salvin SB: Specific depression of delayed hypersensitivity to purified proteins, with relation to production of circulating antibody. Cell Immunol 9~242, 1973. 52. Neta R. Salvin SB: Specific suppression of delayed hypersensitivity. The possible presence of a suppressor B cell in the regulation of delayed hypersensitivity. J lmmunol 113: 1716, 1976. 53. Haynes JD, Askenase PW: Cutaneous basophil responses in neonatal guinea pigs: Active immunization, hapten specific transfer with small amounts of serum and preferential elicitation with phytohemagglutinin skin testing. J lmmunol 118: 1063, 1977. 54. Ovary %: Passive cutaneous anaphylaxis, in Ackroyd JF, editor: Immunological methods. Oxford, 1964, Blackwell Scientific Publications, Ltd., p. 259. 55. Graziano FM, Askenase PW: Involvement of host Fc receptors in antibody mediated cutaneous basophil hypersensitivity reactions. J lmmunol. (In press.) 56. Muller HK, Healy DL: Active cutaneous anaphylaxis in the guinea pig. Immunological and inflammatory reactions. lmmunology 24: 1099, 1973. 57. Parish WE: Eosinophilia. Il. Cutaneous eosinophilia in guinea pigs mediated by passive anaphylaxis with IgG, or reagin, and antigen-antibody complexes; its relation to neutrophils and to mast cells. Immunology 29: 19, 1972. 58. Solley 0. Gleich J, Jordan RE, Schroeter AL: The late phase of the immediate wheal and flare skin reaction. Its dependence upon IgE antibodies. J Clin Invest 58~408, 1976. 59. DeShazo RD, Levinson Al, Dvorak HF. Davis RW: The late phase skin reaction: Evidence for activation of the coagulation system in an IgE-dependent reaction in man. J lmmunol 122:692, 1979. 60. Aakenase PW, DeBernardo R, Tauben D, Kashgarian M: Cutaneous basophil anaphylaxis. Immediate vasopermeability

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increases and anaphylactic degranulation of bahophils at delayed hypersensitivity reactions challenged with additional antigen. Immunology 35:741, 1978. Askenase PW, Tauben D: Unpublished observations. Askenase PW: Immune inflammatory responses to parasites: The role of basophils, mast cells and vasoactive ammes. Am J Trop Med Hyg 26:96, 1977. Plaut M, Lichtenstein LM, Henney CS: Properties 01 a subpopulation of T cells bearing histamine receptors. J Clin Invest 55:856. 1975. Rocklin RE: Modulation of cellular immune responses ire ~‘ivo and in rairro by histamine receptor bearing lymphocytes. J Clin Invest 57: 105 1, 1976. Rocklin RE, Greineder D, Littman BH. Melmon KL: Modulation of cellular immune function in \?rro by histamine receptor-bearing lymphocytes: Mechanism of action. Cell lmmunol 37:162, 1978. Rocklin RE: Histamine-induced suppressor factor (HSF): Effect on migration inhibitory factor (MIF) productton and proliferation. J lmmunol 118: 1734, 1977. Wikel SK. Allen JR: Acquired resistance to ticks. I. Passive transfer of resistance. Immunology 30~3 1 I, 1976. Wikel SK, Allen JR: Acquired resistance to ticks. Il. Effects of cyclophosphamide on resistance. Immunology 30~479. 1976. Gershon RK, Askenase PW, Gershon MD: Requirement for vasoactive amines for production of delayed-type hypersensitivity skin reactions. J Exp Med 143:732, 1975. Schwartz A, Askenase PW, Gershon RK: The effect of locally injected vasoactive amines on the elicitation of delayed-type hypersensitivity. J lmmunol 118: 159. 1977. Uvnas B: Histamine storage and release. Fed Proc 33:2172, 1974. Metzler CM, Askenaae PW, Gershon RK: Dependence on vasoactive amines for the localization of leukocytes in DTH reaction sites and lymph nodes. (Submitted for publication.) Dvorak AM, Mihm MC Jr. Dvorak HF: Morphology of delayed-type hypersensitivity reactions in man. Il. Ultrastructurd1 alterations affecting the microvasculature and the tissue mast cells. Lab Invest 34: 179, 1976. Huber B, Devinsky 0, Gershon RK, Cantor H: Cell mediated immunity. Delayed-type hypersensitivity and cytotoxic responses are mediated by different T cell subclasses. J Exp Med 143: 1534, 1976. Marchesi VT. Gowens JL: The migration of lymphocytes through the endothelium of venules in lymph nodes. An electron microscope study. Proc R Sot Lond [Biol] 159:283, 1964. McCluskey RT, Benacerraf B, McCluskey JW: Studies on the specificity of the cellular infiltrate in delayed hypersensitivity reactions. J lmmunol 58~466, 1976. Lubaroff DM, Waksman BH: Bone marrow as a source of cells in reactions of cellular hypersensitivity. I. Passive transfer of tuberculin sensitivity in syngeneic systems. J Exp Med 128: 1425, 1968. Avella J, Madsen JE, Binder HJ, Askenase PW: Effect of histamine H2-receptor antagonists on delayed hypersensitivity. Lancet 1:624, 1978.