CELLULAR

IMMUNOLOGY

Modulation

37, 162-173 (1978)

of Cellular

Receptor-Bearing

Immune

Function

Lymphocytes:

in Vitro

Mechanism

by Histamine

of Action l

Ross E. ROCKLIN,~ DIRK GREINEDER, BRUCE H. LITTMAN, KENNETH L. MELMON

AND

Department of Medicine, Robert of Clinical Pharmacology,

Cardiovascular

B. Brigham Hospital and Harvard Medical School; Divisiolr and the Departments of Medicine and Pharmacology, Research I&We, University of California School of Medicilze, San Francisco, California 94143 Received

September

22,1977

When soluble histamine is added to guinea pig lymphocytes in vitro, antigen-induced cellular proliferation and the production of migration inhibitory factor is suppressed. The inhibitory effects that are produced by histamine have been shown to be mediated by the histamine-type 2 receptors of the involved cells, but the exact nature of this suppression has not been fully explored. The present studies have evaluated, following immunization, the effect of histamine on macrophage function in vi&o, and affinity chromatography to delete a subpopulation of cells bearing histamine receptors. When we treated monolayers of peritoneal exudate cells with histamine (up to 10e3M) we found that histamine did not interfere with antigen binding by macrophages, macrophage presentation of antigen to lymphocytes, nor the antigen-independent or antigendependent lymphocyte-macrophage rosetting. Columns containing insolubilized conjugates of histamine and rabbit serum albumin depleted a subpopulation of cells responsive to histamine, i.e., the non-adherent cells made migration inhibitory factor and proliferated in the presence of histamine. The latter finding suggested that the retained cells might have suppressor function and if so, might mediate their effect through the release of a soluble factor. Preliminary data obtained in these studies supports this hypothesis. We conclude that cells bearing histamine receptors may serve a regulatory role in cellular immunity after their activation by histamine by producing a non-dialyzable factor with immunosuppressive properties.

INTRODUCTION Histamine has a profound effect on in vitro functions of selected leukocytes. For example, it inhibits release of histamine from basophils that are sensitized by IgE (1)) inhibits release of lysozomal enzymes from neutrophils (Z), and inhibits production or release of antibodies from lymphocytes (3). It can inhibit the cytotoxic effects of some effector T cells (4), and production of migration inhibitory factor (MIF) 3 and antigen stimulated cell proliferation (5, 6). All of these effects 1 Supported in part by United States Public Service Grants AI-11729, GM-16496, HL-06285, and GM-00001. 2 Dr. Rocklin is the recipient of a Research Career Development Award No. 5 K04 A170796. 3 Abbreviations used in this paper : MIF, migration inhibitory factor; AMP, 3’5’ adenosine monophosphate ; OCB-BGG, orthochlorylbenzoyl-bovine gamma globulin ; PEC, peritoneal exudate cells ; LNC, lymph node cells; E, erythrocyte; H, histamine; RSA, rabbit serum albumin. 162 0008-8749/78/0371-0162$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

HISTAMINE

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163

IMMUNITY

are thought to be mediated through the Hz receptor because histamine-type 2 competitive antagonists such as burimamide and metiamide reverse the histamine induced effects at lower concentrations than do Hi antagonists such as chlorpheniramine and mepyramine (2, 5, 7, 8). Stimulation of the Hz receptor is accompanied by an increase in intra-cellular levels of cyclic 3’5’ adenosine monophosphate (AMP) that is thought to mediate at least some of these actions (9). The mechanisms by which histamine accomplishes suppression of immune responses is incompletely understood. The present studies were initiated to examine the way in which histamine affects antigen-induced production of MIF and proliferation of lymphocytes (5). We focused on two questions : (1) Does histamine affect a macrophage-dependent step in the production of MIF ? (2) Can histamine either directly inhibit the production of MIF by lymphocytes or activate a lymphocyte that can modulate the production of MIF by other cells? Our results indicate that histamine does not interfere with the macrophage dependent steps of lymphocyte activation that lead to production of MIF. However, results from studies using affinity columns of insolubilized conjugates of histamine suggest that the lymphocytes that adhere to these columns may play a regulatory role in modulating the proliferation of lymphocytes and their ability to produce MIF. MATERIALS

AND

METHODS

DWJS Histamine hydrochloride (Sigma prepared before each experiment.

Chemical Co., St. Louis,

MO)

was freshly

Animals Male Hartley or strain 2 guinea pigs, approximately 500 g each, were sensitized with 100 pg of orthochlorobenzoyl-bovine gamma globulin (OCB-BGG) in saline that had been emulsified in an equal volume of complete Freund’s adjuvant (Difco Laboratories, Detroit, MI, H37Ra). The antigen-adjuvant emulsion (l/10 ml) was injected into each footpad (10). Direct MIF

Test

Peritoneal exudate cells (PEC) f rom OCB-BGG immune guinea pigs were harvested according to the method of David and David (10). Exudates were induced 2 weeks after sensitization. The PEC were packed into capillary tubes and (conplaced in Mackaness-type chambers. Tissue culture medium (TC-199) taining 15% normal guinea pig serum) alone or containing varying concentrations of OCB-BGG (0.01-10.0 pg/ml), with or without additional drugs, were used to fill the chambers. The chambers were incubated at 37°C for 18 hr, at which time the area of migration was drawn, determined by planimetry, and the percent of migration inhibition was calculated by the following formula : % migration inhibition area of migration area of migration

in presence of antigen and/or in absence of antigen and/or

drug drug

1 > x 100

164 Indirect MIF

ROCKLIN

ET

AL.

Test

Two to four weeks after immunization, lymph node cells (LNC) were obtained from OCB-BGG immune guinea pigs with the use of the method of David and David (10). The popliteal, axillary and inguinal lymph nodes were teased with the use of a mouse tooth forceps. The sediment was discarded and the cells were washed twice in medium TC-199 that contained 100 U of penicillin/ml and 100 pg of streptomycin/ml. The LNC were then layered onto Ficoll-Hypaque gradients (LSM, Litton Bionetics, Bethesda, MD) and centrifuged 400s for 40 min at room temperature. The interface cells were collected, washed two times and viability (> 90%) was determined by trypan blue exclusion. The final cell concentration was adjusted to 10 X lo6 cells/ml in medium TC-199 without serum. The cell suspension was divided into two aliquots; to one aliquot, 100 pg/ml OCB-BGG was added and to the other the same volume of saline was added. Varying concentrations of drug were added along with antigen. The cell suspensions were incubated for 24 hr at 37°C in a 5% CO*-957 o air atmosphere. The cell-free supernates were obtained by centrifugation and made to contain 15% guinea pig serum by volume. These supernates were assayed for MIF activity ; normal guinea pig PEC in capillary tubes were used, as described above. The percent migration inhibition was calculated with the use of the above formula. Lyfnphocyte Proliferation Lymph node lymphocytes from OCB-BGG immune animals were cultured in vitro for [3H] thymidine incorporation (11). Two x lo5 cells per well were cultured in microtiter plates in TC-199 that contained 15% normal guinea pig serum and 50 pg/ml OCB-BGG with or without drug for 3 days at 37°C in a 5% COz95% air atmosphere. One microcurie of [3H] thymidine (Sp. Act. 6.7 &i/M, New England Nuclear, Boston, MA) was added to each well 18 hr before terminating the cultures. The cells were harvested (MASH II, Microbiological Associates, Bethesda, MD) and the radioactivity was determined by scintillation counting. The mean counts per minute (CPM) of quadruplicate cultures were recorded and a stimulation index was calculated from the ratio of cpm of antigen-stimulated cultures/cpm of unstimulated cultures. The effect of histamine on antigen-induced lymphocyte proliferation was compared to untreated cultures. Antigen Binding of Macrofihages Oil-induced PEC (5 X lo6 cells/ml) from normal strain 2 guinea pigs were plated for 1 hr at 37°C and washed free of nonadherent cells. The macrophage monolayers were then either pretreated with lo+ M histamine or saline for 30 min at 37°C prior to being pulsed with antigen, or histamine and antigen were added simultaneously. OCB-BGG (100 &ml) was added to monolayers for 30 min and then they were washed four times with medium TC-199. LNC were obtained from OCB-BGG immune strain 2 guinea pigs and passed twice over nylonwool columns to deplete them of adherent cells (12). Such preparations were incapable of producing MIF or proliferation unless adherent cells were added back to the cultures. These nonadherent LNC were cultured over the treated monolayers. No further antigen was added to the cultures, which were allowed to incubate for

HISTAMINE

EFFECT

ON

CELLULAR

IMMUNITY

16.5

18 hr at 37°C in a 5% COZ-95(r 0 air atmosphere. The cell-free supernatants were harvested and assayed for MIF activity. Macrophage-Lymphocyte

Binding Assay

The macrophage-lymphocyte binding assay was performed as previously described (13). Oil-induced PEC were plated overnight in slide chambers (Lab-Tek Products, Westmount, IL), and washed to obtain macrophage monolayers. Histamine ( 10m3iV>, Tuberculin PPD (2.5 /*g/ml) or phosphate buffered saline (PBS) was added to appropriate chambers, followed by 5 to 6~ 10GLNC in TC-199 containing 10% normal guinea pig serum. Cultures were incubated with slow rocking at 37°C in a humidified atmosphere of 95% air and 5% COP. At the appropriate times, cultures were washed to remove nonadherent cells, fixed with 1% gluteraldehyde and stained with acetone-Giemsa. Lymphocyte binding per 100 randomly selected macrophages was quantitated by visual counting. Enumeration

of T Cells

Rabbit erythrocyte rosette binding lymphocytes (E-rosetted cells). Fresh heparinized rabbit blood was washed three times with cold Hanks balanced salt solution and adjusted to 0.5% concentration. One hundred microliters of this rabbit erythrocyte (E) suspension, 100 ~1 of lymphocyte suspension at 5 x lo6 cells/ml and 20 ~1 of guinea pig serum previously absorbed twice with rabbit erythrocytes were combined in 6 x 50 mm glass culture tubes (14). These were incubated at 37°C for 5 min, centrifuged at 409 for 5 min at 4”C, and incubated in an ice water bath for 1 hr. Cells were gently resuspended with a Pasteur pipette and placed on a glass slide that had previously been coated with toluidine blue. Lymphocytes were counted and categorized as not binding E, binding 1-2E/lymphocyte, or binding 2 3 E/lymphocyte. I%agnunofluorescent Test for Surface Immunoglobulin

Bearing Lymphocytes

A lymphocyte suspension (0.4 ml) containing 5 x lo6 cells/ml was centrifuged at 18Og for 10 min at 4”C, and 0.3 ml of the cell-free supernatant was removed. Fluorescein conjugated IgG fraction of rabbit anti-guinea pig gamma globulin (Cappel Laboratories, Dunnington, PA) (100 X) was added to the cell pellet, mixed with a Pasteur pipette and incubated 30 min at 37°C with periodic agitation. Ten percent fetal calf serum in Hanks balanced salt solution was added to the cells which were washed three times by centrifugation at 18Og for 10 min at 4°C. The cells were resuspended in fresh cold Hanks balanced salt solution. The percentage of Ig bearing lymphocytes was then determined by immunofluorescent and light microscopy. Histamine-Sepharose

Chromatography

Affinity columns that contained insolubilized histamine were prepared as previously described (15, 16). Histamine (H) was covalently bound to rabbit serum albumin (RSA) via carbodiimide and this hormone conjugate in turn was coupled to cyanogen bromide-activated Sepharose (H-RSA) . Control columns consisted of RSA alone bound to Sepharose or histidine methyl ester conjugated to RSA-

166

ROCKLIN

AL.

ET

TABLE

1

Effect of Histamine on Antigen Binding of Macrophages MIF Activity (% Migration Inhibition) Experiment

Pre-Treatment” Control

Histamine

No Pre-Treatment6 Control

(1OF M)

1 2 3

48 20 48

44 23 42

Histamine (lo+

47 33 35

M)

47 41 34

a Strain 2 guinea pig macrophage monolayers pretreated with low3 M histamine for 30 min at 37°C and then pulsed with OCB-BGG (100 pg/ml) for another 30 min at 37°C. The plates were washed three times in medium TC-199 and then lymphocytes added. b 10-S M histamine and OCB-BGG (100 pg/ m I) were added simultaneously to strain 2 guinea pig macrophage monolayers for 30 min at 37% The plates were washed and then lymphocytes added.

Sepharose. LNC were obtained from OCB-BGG immune animals and centrifuged on Ficoll-Hypaque,gradients (400g) for 40 min at room temperature. The interface cells ( > 957 o viable) were washed three times in medium TC-199 and resuspended to 100 x lo6 cells/ml in serum free medium. One ml aliquots of cells were incubated with 0.6 ml of a 25% suspension of Sepharose beads for 15 min at 37°C. The mixture was then carefully pipetted into a 10 ml pipette that was plugged with nylon wool. The columns were washed with 1 ml of medium and the nonadherent cells collected. Attempts were made to elute the adherent cells with akaline (pH8) medium TC-199 or free histamine. The nonadherent cells were washed, adjusted to 10 x lo6 cells/ml, and tested for production of MIF with or without added histamine. Generation and Assay of a Histamine Induced Suppressor Factor (HSF) 10 X lo6 LNC/ml were exposed in vitro to varying concentrations of histamine in TC-199 (containing 15% normal guinea pig serum) to determine whether a soluble factor would be released. An assumption was made initially that the hypothetical factor would not be dialyzable, so that histamine could be removed from the supernatant. The cells were cultured for 24 hr at 37°C in the absence or presence of histamine ( 10-3-10-6 JJ) and the cell-free supernatants obtained by centrifugation. Histamine ( 10m3M) was added to the control supernatant prior to dialyzing the supernatants against 0.15 N saline for 18 hr and redialyzed against fresh TC-199 for 6 hr. The supernatants were sterilized by Millipore filtration and assayed for their effect on MIF production as described above. RESULTS Eflect of Histamine on Macrophage-Lymphocyte

Interaction

Macrophages were assessed for their ability to bind antigen for presentation to LNC after being treated with histamine (Table 1). Macrophages were either pretreated with histamine and then pulsed with antigen or histamine and antigen were added together. As can be seen in the three experiments carried out under both

HISTAMINE

EFFECT

ON

TABLE Failure Expt. numbera

of Histamine

Time (hr)

to Inhibit

-

CELLULAR

2

Macrophage-Lymphocyte

Lymphocytes

bound/100

Rosette

l-3 ‘4 5

+PPD

Histamine (10-Z M)

P

1

53.1 f

4.6

58.8 f

3.5

NS

7.1 f

1.6

7.2 f

1.0

NS

1

117.4 f

4.2

122.4 f

3.2

NS

2.5

NS

17.2 f

3.1

14.0 f

No

Histamine (10-Z M)

treatment

20 20

Formation

macrophages

-PPD No treatment

167

IMMUNITY

37.6 f

2.5

37.1 f

6.2

110.7 f

102.4 f

P -

3.4

NS

8.6

NS

-

-

a Experiments l-3 were performed with nylon column purified lymphocyte whereas experiments 4 and 5 were performed with Ficol-Hypaque purified lymphocytes. Each experimental point consists of duplicate slides, counted twice. The results are expressed as mean lymphocytes bound/100 macrophages f 1 SEM.

conditions, histamine-treated macrophages were able to bind antigen and to present it to LNC that were depleted of macrophages. Production of MIF was not significantly different in either case. We also determined whether histamine would affect lymphocyte-macrophage rosette formation in vitro. This interaction is non-specific (antigen-independent) at 1 hr and antigen specific at 20 hr. Macrophage monolayers were pretreated with histamine and LNC (depleted of macrophages) were added with or without antigen. The number of lymphocytes rosetting around macrophages were counted at 1 hr and 20 hr. As shown in Table 2, histamine did not interfere with either nonspecific or antigen-specific rosetting. Afinity

Chrowtatography

z&h Histamine-Sepharose

H-RSA Sepharose columns have been used to deplete populations of heterogeneous human leukocytes or murine splenic lymphocytes of those cells that bear receptors for histamine. The others pass through the column and do not increase their content of cyclic AMP after exposure to histamine (17). Furthermore, the binding of cells to the columns could be blocked either by soluble histamine or by antihistamines (18). In the experiments reported here, H-RSA columns consistently bound more cells than the RSA columns, the average yield for 12 experiments being 72% for the former and 65% for the latter. The numbers of T cells (E-rosette positive) and B cells (Ig+) were quantitated after they passed over the columns. The results in Table 3 indicate that the H-RSA and RSA columns bind B cells to the same extent. E-rosette positive cells (> 3 RBC/lymphocyte) are increased equivalently by both columns. Unseparated OCB-BGG immune LNC, and those that were not adherent to H-RSA and RSA columns, were tested for their ability to produce MIF and proliferate in response to specific antigen in the presence and absence of histamine. The results shown in Fig. 1 indicate that in the absence of histamine all three populations produced equivalent amounts of MIF to OCB-BGG. However, in the presence of histamine only the cells that had passed through H-RSA columns were

168

ROCKLIN

-10

COll~~Ol

ET

AL.

IO-‘M ~Histamine

-TL

FIG. 1. Removal of cells sensitive to histamine by affinity chromatography. Histamine-rabbit serum albumin conjugates (H-RSA) were bound to activated sepharose. Control column consisted of RSA alone bound to sepharose. Immune LNC were passed over the column and the non-adherent cells were tested for their ability to make MIF in the presence and absence of histamine. Cells that were not adherent to H-RSA, but not those that passed through RSA or that were unfractionated, were able to produce MIF when cultured with histamine.

able to make MIF. These results are consistent with the removal of a histamine sensitive population by the H-RSA column or with nonspecific effects of the column on nonadherent cells that made them unresponsive to histamine. In order to distinguish between these two possibilities, the experiments were repeated and the cells that had passed through the columns were divided into two aliquots. One group was tested as before and the other aliquot was allowed to “recover” for 18 hr and then tested for production of MIF. In the three experiments summarized in Fig. 2 essentially the same results were obtained, i.e., in the presence of histamine, cells allowed to recover for 18 hr still produce MIF to the same extent as they had before the recovery period. Furthermore, in recent studies we have been able to demonstrate that human leukocytes and murine splenic lymphocytes that were not TABLE

3

Numbers of E-Rosette Positive and Ig Positive Cells Following Passage Over Histamine Columns E-Rosettes (‘%)

Cells

Total

% Ig + (immunofluorescence)

23

1-2

28 54

34 12

62 66

34 24

54 72

14 10

68 82

23

2

RSA Expt. 1

44

23

67

26

2

71

10

81

16

Unseparated Expt. 1 2

H-RSA Expt. 1

15

HISTAMINE

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FIG. 2. Immune LNC were chromatographed on H-RSA and RSA columns and either tested immediately (left panel) for their sensitivity to histamine or allowed to “recover” for 18 hr (right panel) before exposing them to histamine. Cells allowed to recover for 18 hr still produced MIF in the presence of histamine indicating that histamine receptor-bearing lymphocytes were retained on the H-RSA column and did not pass through in a desensitized state.

adherent to the H-RSA column were able to respond as well by increasing their adenylate cyclase to cholera toxin as the same number of unchromatographed cells or cells that had passed through RSA columns (17). Thus, they were not separated by their simple complement of membrane cyclase nor were they nonspecifically but sub-lethally damaged. The results of six experiments depicting antigen-induced proliferation of unfractionated cells and those that had passed through columns are presented in Fig. 3. In the absence of histamine, all three populations proliferated similarly to OCBBGG. However, as was seen with production of MIF, only the cells that did not adhere to H-RSA columns were capable of normal proliferation in the presence of histamine. The columns did not consistently alter the baseline incorporation of

lo%

CONTROL -HISTAMINE’

3. Column passed cells were tested for antigen-induced proliferation in the presence of histamine. H-RSA passed cells treated with histamine proliferate to the same extent as untreated cells. RSA passed and unfractionated cells have their proliferative response suppressed by histamine. FIG.

170

ROCKLIN

COllld

IO-%4

ET

lO+M

AL.

10-6~

-H”TAMfNf--FIG. 4. Effect of histamine induced suppressor factor (HSF) on guinea pig MIF production. Supernatants derived from 10 X 10” LNC/ml cultured with or without varying concentrations of histamine (lo-* to lo+ M) for 24 hr at 37°C. Supernatants dialyzed against normal saline for 18 hr and redialyzed against fresh TC-199 for 6 hr. These supernatants were then used to culture OCB-BGG immune LNC for MIF production.

[3H] thymidine. Similar results were obtained with production of MIF and cell proliferation when another antigen (Tuberculin PPD) was used. Furthermore, histidine methyl ester-RSA Sepharose columns gave results that were similar to the RSA controls. The viability of cell populations not adhering to H-RSA and RSA columns was similar. Efect

of Histamine Suppressor Factor (HSF)

on MIF Production

Supernatants obtained from immune LNC stimulated by histamine were used as culture media to resuspend fresh immune LNC. The latter cells were then exposed to specific antigen and the effect of these supernatants on MIF production was observed. The results of 3 experiments are summarized in Fig. 4. It may be seen that supernatants obtained from LNC stimulated by 10d3 to 1O-5 M histamine significantly (P < 0.01) suppressed MIF production compared to the control supernatant. DISCUSSION Our approach to studying the mechanism of histamine suppression of MIF and cellular proliferation has been twofold. Previous work suggested that histamine could affect a macrophage-dependent stage in MIF production and/or activate some cell that could regulate lymphocyte function (5). We have, therefore, used two assays that evaluate lymphocyte-macrophage interaction to study the former columns for the latter. The results possibility and histamine-RSA-Sepharose reported here suggest that histamine may act on a regulatory cell that modulates production of MIF and cell proliferation. The evidence for this conclusion will be discussed below. Of particular interest, our studies have revealed that histamine stimulates the production of a soluble suppressor factor that may mediate this effect. There is now substantial evidence to support the notion that lymphocyte-macrophage interaction is required for lymphocyte activation to occur and ultimately leads to production of mediator and cell proliferation (21-25). While previous reports have indicated that macrophages do not have receptors for histamine (5, 26), it is necessary to rule out the possibility that histamine might interfere with a macrophage-dependent step in production of MIF. This was studied in two ways.

HISTAMINE

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171

One approach was to “pulse” nonimmune macrophages that had been pretreated with histamine with antigen or to add histamine concommitantly with the antigen. These macrophage monolayers were then washed and immune LNC (depleted of macrophages) were added. No further antigen was introduced into this system. The question being asked here was whether histamine interfered in any way with the macrophage binding of antigen such that these cells were no longer able to present sufficient amounts of antigen to immune lymphocytes for activation to occur. The results of these experiments indicate that histamine did not prevent antigen binding or antigen presentation to immune LNC by macrophages (Table 2). The other way we chose to examine lymphocyte-macrophage interaction was to determine whether histamine interfered with lymphocyte-macrophage rosetting. The latter phenomenon was described as involving two phases: A non-specific (antigen independent) rosetting of lymphocytes about macrophages measured at 1 hr and an antigen-specific phase measured at 20 hr (13, 27). The latter binding required Ia compatible lymphocytes and macrophages while the former does not (27). Moreover, increased antigen-specific lymphocyte-macrophage rosetting is associated with a positive proliferative response to the same antigen (27). In our experiments, histamine did not suppress either the nonspecific or antigen-specific rosetting response (Table 3). Taken together, the accumulated evidence would indicate that histamine itself does not operate at the level of the macrophage. However, this does not rule out the macrophage as the site of action of the suppressor factor, whose secretion may be stimulated by histamine (described below). Another approach taken to study the mechanism of suppression by histamine involved the use of Sepharose columns that contained insolubilized conjugates of histamine to RSA (H-RSA) . Such columns remove cells that bear specific amine receptors (16, 28). The mechanism of binding cells to the H-RSA columns is not clear, although present evidence would indicate that it is related to a specific function of the histamine moiety of the conjugate because the binding can be blocked by histamine and antihistamines and the passed cells do not respond to soluble histamine by raising their levels of cyclic AMP (17). Passage of immune LNC over H-RSA columns rendered the nonadherent cells insensitive to histamine, i.e., these cells made MIF and proliferated in the presence of histamine to the same extent as untreated cells (Figs. 1 and 3). Cells that passed through the control columns (RSA or histidine methyl ester-RSA columns) retained their sensitivity to histamine. That cells bearing histamine receptors were actually removed by H-RSA columns and were not present in a “desensitized” or unresponsive state to histamine was suggested by experiments in which cells that were not adherent to columns were allowed to “recover” for 18 hr prior to being stimulated to make MIF (Fig. 2). In these experiments, H-RSA passed cells did not regain their sensitivity to histamine. If these cells were present but desensitized, they should have regained their responsiveness to histamine within that time. Evidence in favor of the latter conclusion is provided by the following observations : ( 1) Other hormone receptors such as insulin and beta-adrenergic receptors which have been reduced in number by proteolysis or desensitization reappear within 18 hr (2% 30) ; (2) tvp sin-treated lymphocytes fail to produce a suppressor factor immediately following a histamine pluse but regain their ability to do so after 18 hr (unpublished data) ; (3) lymphocytes chromatographed on H-RSA columns do not make histamine suppressor factor but the retained cells do (Rocklin, Greineder

172

ROCKLIN

ET

AL.

and Melmon, manuscript in preparation). Furthermore, H-RSA columns removed on the average 7% more cells than did RSA columns, Unfortunately, the bound cells that were responsive to histamine could not be eluted from the columns. The results described above imply that a regulatory cell bearing j histamine receptor was being removed by passage over H-RSA columns. This interpretation was chosen because cells capable of production of MIF and of proliferation did not ahhere to the columns and therefore did not appear to have receptors for histamine. If the histamine receptbr-bearing cell serves a regulatory function, that function might be mediated by a soluble product. In fact, this appears to be the case (Fig. 4). LNC stimulated by histamine for 24 hr elaborate a non-dialyzable (2.?-40,000 daltons) factor into the cell-free supernatant, which, when cultured with fresh LNC, reversibly suppresses the production of MIF and cell proliferation (20). Production of the suppressor factor can be blocked by Hz-receptor antagonists but not HI-receptor antagonists. The nature of this factor is currently being explored. In the mouse, the mechanism of action of histamine on immune responses appears to differ from that described here in the guinea pig. Lymphocyte-mediated cytotoxicity in vitro is inhibited by histamine (4, 8). However, in this instance, it has been proposed that the cytotoxic cells are the ones that bear histamine receptors and are “turned off” by an effect of histamine that increases intracellular levels of cyclic AMP (1, 9). Investigators have used an antibody system in mice to show that histamine receptor-bearing cells have a regulatory role (3). When these cells are removed by H-RSA columns, antibody production increases. It has been proposed that the regulatory cells involved in this process continually make suppressor material and the process is halted by the addition of histamine. Here again, histamine stimulates the accummulation of intracellular cyclic AMP, which would “turn off” the suppressor cell. The latter interpretation of the humoral antibody response contrasts with the findings reported here. The present data suggest that histamine activates an as yet unidentified suppressor cell to produce a soluble factor that modulates cellular-immune function. At present, we would tend to invoke two types of suppressor cells responsive to histamine, one whose function is inhibited and another whose function is augmented by the amine. In this way, the presence of histamine would enhance antibody production and suppress cellular immunity or its absence would increase the “tone” in the alternative direction. ACKNOWLEDGMENTS The authors wish to acknowledge the excellent technical assistance of Ms. Hollis Dolben and Ms. Barbara Sherry.

REFERENCES 1. Bourne, H. R., Melmon, K. L., and Lichtenstein, L. M., Science (Washington, D.C.) 173, 1303, 1971. 2. Weissman, G., Zurier, R. B., and Hoffstein, S., In “Cyclic Nucleotides, Immune Response and Tumor Growth” (W. Braun, C. Parker, and L. M. Lichtenstein, Eds.), pp. 176-189. New York Academic Press, Inc., New York, 1974. 3. Shearer, G. M., Melmon, K. L., Weinstein, Y., and Sela, M., J. Exp. Med. 136, 1302, 1972. 4. Henney, C. S., Bourne, H. R., and Lichtenstein, L. M., Z. Zmmzirzol. 108, 1526, 1972. 5. Rocklin, R. E., J. Clin. Znvesf. 57, 1051, 1976 6. Ballett, J. J., and Merler, E., Cell. Zmmunol. 24, 250, 1976.

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Modulation of cellular immune function in vitro by histamine receptor-bearing lymphocytes: mechanism of action.

CELLULAR IMMUNOLOGY Modulation 37, 162-173 (1978) of Cellular Receptor-Bearing Immune Function Lymphocytes: in Vitro Mechanism by Histamine...
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