Histamine modulates in vitro IgG production by pokeweed mitogen-stimulated human mononuclear cells

CELLULAR

IMMUNOLOGY

64,

324-336 (1981)

Histamine Modulates in Vitro IgG Production by Pokeweed Mitogen-Stimulated Human Mononuclear Cells’ MARCONDES LIMA’

AND

Ross E. ROCKLIN

Division of Allergy and Immunology, Department of Medicine, Tufts University School of Medicine, New England Medical Center Hospital, Boston, Massachusetts 02111 Received April 17. 1981; accepted August 19, 1981 The modulatory role of histamine was investigated in an in vitro model involving immunoglobulin production by human blood mononuclear cells. The addition of histamine in vitro had little effect on the spontaneous production of IgG (measured by double-antibody radioimmunoassay). However, the addition of histamine ( 10-4-10-9M) to mononuclear cells stimulated with an optimun concentration of pokeweed mitogen (PWM) significantly suppressed IgG production in a dose-dependent fashion from 10m4to 10m6iU (range 55-37% suppressor, respectively). The kinetics of histamine-induced suppression was investigated by adding histamine to mononuclear cells at the time of stimulation by PWM or up to 4 days later. Histamine only significantly suppressed PWM induced IgG synthesis if added up to 72 hr after the addition of PWM. In order to characterize the histamine-responsive suppressor cell in this system we employed a coculture technique and compared our findings to those of Con A-induced suppression. Mononuclear cells were either separated into E-rosette-positive (T-enriched) or E-rosettenegative (T-depleted) populations, irradiated, or treated with monoclonal antibodies plus complement; cells were then stimulated with histamine or Con A for 24 hr and cocultured with autologous E-rosette-negative populations that were then stimulated with PWM, and IgG production was measured. The histamine- and Con A-responsive suppressor cell was found within the T-enriched population, was radiation sensitive to 2000 and 3000 rad, respectively, and was identified by OKT8 antisera but not OKT4 antisera. Of particular interest, the addition of histamine to irradiated T cells and PWM-stimulated T-depleted populations did not result in suppression of IgG production, implying that B cells were not being inhibited directly by histamine. The nature of the histamine receptor present on the suppressor cell was investigated. The H, antagonist, diphenhydramine (lo-“ M), inhibited IgG production as effectively as histamine. The combinations of H, antagonist and histamine did not result in an additive effect. Cimetidine ( 10s4M), had no effect by itself on in vitro IgG production and the combination of cimetidine plus histamine in equimolar concentrations did not abrogate histamine-induced inhibition of IgG production. Studies employing the HI and H2 agonists ( tom4to low6 M), 2-pyridylethylamine and Dimaprit, respectively, resulted in an equivalent suppression of IgG production. The H, and HZ agonist-responsive suppressor cell was found in the T-enriched population and was sensitive to 3000 rad. These experiments imply that the histamine-reactive cell suppressing IgG synthesis may be activated through either its HI or HZ receptor. ’ Supported by USPHS Grant AI- 16312. * Supported by a grant from CAPES of Brazil. 324 0008-8749/81/160324-13$02.00/O Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form nscrved

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INTRODUCTION The ability of histamine to influence the inflammatory process by modulating a variety of polymorphonuclear leukocyte and lymphocyte functions is well established and its role in regulating certain immune responses,in particular, has become an area of intense investigation. Thus far, the effects of histamine on lymphocyte function have been studied in greater detail in systems that employ T-cell assays. For example, it has been shown that histamine directly inhibits cell-mediated cytolysis by sensitized T cells through a mechanism which appears to involve cyclic AMP (1, 2). On the other hand, such T-cell functions as lymphocyte proliferation to mitogens and antigens and lymphokine production are also inhibited by histamine but possibly through another mechanism (3-7). The latter involves the activation of a histamine receptor bearing suppressor T cell by histamine which then results in the production of a soluble suppressor factor (histamine-induced suppressor factor or HSF3). HSF may inhibit the proliferating T cells directly, or more likely, work through an indirect mechanism involving monocytes and prostaglandin synthesis (8). The histamine-induced suppressor cell, as well as the suppression of Tcell-mediated cytolysis, involves stimulation of the histamine type 2 receptor (2-7). Regulation of B-cell responses by histamine has been less well studied. Shearer and co-workers have previously shown that plaque-forming cell numbers may be increased if murine splenic lymphocytes are first chromatographed on columns containing insolubilized histamine (9). Furthermore, histamine may suppress plaque formation in vitro when spleen cells are cultured in the presence of the drug ( 10, 11). Employing a model of tolerance involving SJL/J mouse spleen cells made unresponsive to the multichain synthetic peptide (T,G)-Pro-L, Moses et al. showed that prior incubation of these spleen cells with histamine abrogated the suppression ( 12). Little information has been accumulated thus far concerning immunoregulation of humoral responses by histamine in man. In the present study, we have investigated the effects of histamine on human IgG production in vitro using a double-antibody radioimmunoassay. Our results demonstrate that histamine, in a dose-dependent fashion, suppresseshuman IgG production generated by pokeweed mitogen (PWM) in vitro. Furthermore, the histamine-responsive suppressor cell is contained within the E-rosette-positive population, bears on its surface a differentiation antigen identified by OKT8+ antisera, is radiation sensitive (2000 rad), and is probably activated through either its histamine type 1 or type 2 receptor. MATERIALS

AND METHODS

Reagents. Pokeweed mitogen (Grand Island Biological Co., Grand Island, N.Y.) was dissolved in 5 ml of sterile distilled water and then diluted 1:10 with medium TC-199. Concanavalin A (Miles Yeda, Elkhart, Ind.) was dissolved in TC-199 at a concentration of 1 mg/ml. H, (diphenhydramine) and Hz (cimetidine) antagonists and Hr (2-pyridylethylamine) and Hz (dimaprit) agonists were kindly supplied by Smith, Kline, and French, Philadelphia. Monoclonal antibodies (OKT4 and OKT8) against T-cell subsets were kindly supplied by Ortho Pharmaceutical Corporation, Raritan, New Jersey. 3 Abbreviations used: Con A, concanavalin A; HSF, histamine-induced suppressor factor; PWM, pokeweed mitogen.

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Production of supernatants containing ZgG by human lymphocytes. Heparinized venous blood was drawn from normal donors after obtaining informed consent. The mononuclear cells were isolated on Ficoll-Hypaque density gradients (13). Cells at the interface were harvested and washed four times with medium TC-199 containing 5% heat-inactivated fetal calf serum. The cell concentration was adjusted to 2 X lo6 mononuclear cells/ml in medium TC-199 containing 10% fetal calf serum, 25 mM Hepes buffer, 100 units/ml penicillin, and 100 @g/ml streptomycin. Two-milliliter cultures were incubated in 12 X 75-mm plastic tubes (Falcon Plastics) at 37°C in a 5% CO*-95% humidified air atmosphere for 7 days. At the end of the incubation period, the cell-free supernatants were harvested following centrifugation and were assayed for their content of IgG antibody. Assay of ZgG. IgG was measured by the use of a double-antibody radioimmunoassay (14). Purified IgG was obtained from Cappel Laboratories (Cochranville, Pa) and further purified by gel filtration on a Sephadex G- 150 column. Goat antihuman IgG (Cappel Laboratories) was absorbed with free K and X light chains (Cappel Laboratories) to render them specific for y chains. Specificities of the antiserums were confirmed by Ouchterlony double-diffusion tests. IgG was iodinated by the Chloramine-T method (15). More than 98% of the I’25 bound to IgG was precipitable with trichloroacetic acid. IgG content in the supernatants of the cultured mononuclear cells was measured by use of a double-antibody radioimmunoassay performed in microtiter wells (Microtiter Plates, Gibco) with rabbit anti-human IgG (first antibody), I ‘25-labeled IgG, and a goat anti-rabbit IgG as the second antibody. The techniques used to define the specificity and accuracy of the tests were identical to those described for double-antibody radioimmunoassays of other immunoglobulins ( 16). Preparation of T-enriched and non-T-cell populations by E-rosette formation. Human blood mononuclear cells that were isolated by Ficoll-Hypaque density gradient centrifugation were incubated with neuraminidase (Vibrio choZera)treated sheep red blood cells (0.5 ml SRBC:0.5 ml mononuclear cells at 10 X 10e6 cells/ml) as previously described (17). Following a 2-hr incubation at 4°C the rosettes were gently resuspended and overlaid on a Ficoll-Hypaque gradient (4 ml cell suspension:3 ml Ficoll-Hypaque). The cells were centrifuged (400g) and the E-rosette-negative or non-T cells (interface layer) and the E-rosette-positive or Tenriched cells (pellet) were recovered. The sheep red blood cells were lysed by treatment with ammonium chloride. Following the latter procedure, the T-enriched cells were washed three times in medium TC-199 and resuspended in their appropriate cell concentration. In some experiments, T-enriched cells or E-rosette-negative cells were initially cultured with histamine and/or either H, or Hz antagonists or agonists alone for 24 hr. These cells were then washed twice and cocultured with autologous E-rosette-negative cells. The latter cell mixture was then stimulated with pokeweed mitogen and IgG content of the supernatant was measured. The purity of the E-rosette-positive and -negative populations was determined. The Erosette-positive population contained 92-98% E-rosette-“positive” cells and the Erosette-negative population was contaminated by 2-8% E-rosette-“positive” cells. Characterization of suppressor cells by radiation and monoclonal antisera. TEnriched cells were irradiated (cesium source) with a dose of 1000-3000 rad. These cells and nonirradiated E-rosette-positive cells were then analyzed for their ability to function as suppressor cells following a 24-hr preincubation with histamine or

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Con A. The latter cells were cocultured with autologous E-rosette-negative indicator cells. E-Rosette-positive cells were also treated with OKT4 and OKT8 antisera (5 ~1antibody/10e6 cells) for 30 min at 37”C, washed three times, and then treated with guinea pig serum as a source of complement (1:3 by volume) for 1 hr at 37°C (18). The cells were washed three times and the viability of the remaining cells was determined by trypan blue exclusion. In general, treatment of T-enriched cells with OKT4 antisera plus complement lysed approximately 40-60% of the cells while treatment with OKT8 plus complement caused the lysis of lo-20% of the cells. E-Rosette-positive cells treated with the above monoclonal antibodies were then evaluated for their ability to mediate histamine- or Con A-induced suppression of IgG production when cocultured with autologous E-rosette-negative cells stimulated with PWM. RESULTS Effect on IgG Production of the Direct Addition clear Cells

of Histamine to Mononu-

Our preliminary studies culturing human mononuclear cells for the detection of IgG in vitro indicated that the optimum concentration of PWM was 100 pi/ml cells and that 7 days of culture was also optimum for IgG production. Mononuclear cells from normal subjects were cultured with and without PWM and varying concentrations of histamine ( lop4 and lop9 M). The IgG content of the supernatants was assayed using double-antibody radioimmunoassay. The results of nine experiments are summarized in Fig. 1. PWM induced almost a mean fivefold increase in IgG production compared to the amount that was spontaneously released. In the presence of histamine, PWM-induced IgG content of supernatants was significantly reduced (P < 0.01) between 10e4and low6 M. This corresponded to a 55% ( lop4 M) to 37% ( 10e6 M) reduction in IgG production. Histamine had no significant effect upon the spontaneous production of IgG in the cultures. These ex-

HistomlneW

-

-

10M4

IO*

66

o-7

10-a

FIG. 1. Suppression of in vitro IgG production by the direct addition of histamine to mononuclear cells from normal subjects stimulated by PWM. Data expressed as rig/ml IgG (0 0) and percentage suppression of IgG (0 - - - 0). Mean (&SD) of nine experiments.

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LIMA AND ROCKLIN

periments do not distinguish whether histamine is inhibiting synthesis and/or secretion of IgG. The kinetics of inhibition by histamine was then examined. Mononuclear cells were cultured in the presence of pokeweed mitogen and then histamine was added at the same time or at 24-hr intervals thereafter for up to 96 hr. The results of four experiments are summarized in Fig. 2. Histamine added at time zero caused significant suppression of IgG at all three concentrations used ( 10e4 to 10e6 M). When histamine was added at 24, 48, 72, and 96 hr after the addition of PWM, there was a time-dependent reduction in the amount of suppression observed. At 10e4 M, histamine added at 72 hr after the addition of PWM still induced 20% suppression of IgG synthesis. However, with lower concentrations of histamine, suppression was not significant if the addition of the drug was delayed until 24 hr after the addition of PWM. Characterization of the Histamine-Responsive Cell Using a Coculture Technique In order to partially characterize the histamine-responsive suppressor cell, a coculture technique was employed in which the “suppressor” cell was preactivated with histamine (or Con A for purposes of comparison) for 24 hr, washed, and then combined with autologous E-rosette-negative cells. The putative suppressor cell populations were either divided into E-rosette-positive and E-rosette-negative cells, treated with monoclonal antibodies, or irradiated prior to their incubation with histamine or Con A. T-Enriched or non-T cells were initially cultured in the presence of histamine ( 10m4or 10e5M) or Con A (5 rg/ml) for 24 hr. These cells were then washed and cocultured in a 1:1 ratio with autologous E-rosette-negative cells that were stimulated with PWM, and IgG production was measured. The results of three experiments are summarized in Fig. 3. Both the unfractionated and E-rosette-positive suppressor cell populations accounted for virtually all of the suppression due to HISTAMINE

0 Tim

CONC. (Ml

-.-

10-4

--.--

10-5

24 48 72 96 of Addition of Histanina hs)

FIG. 2. Kinetic study of histamine-induced suppression of IgG production. Human mononuclear cells were stimulated with PWM and histamine was added at the same time or at 24-hr intervals thereafter. Histamine (lo-’ M) suppressed IgG up to 72 hr after the addition of PWM.

HISTAMINE

MODULATES

IgG PRODUCTION

SUPPRESSOR

329

CELLS

UNFRACTIONATED El E - ROSETTE

$

E -ROSETTE

8

I 100

10-4

10-s

Histamine(M)

5 ug/ml ConA

FIG. 3. Histamine- and Con A-induced suppression of IgG by T-enriched and non-T cells using a coculture technique. Unfractionated, E-rosette-positive, or E-rosette-negative populations were preincubated with histamine or Con A, washed, and cocultured with autologous E-rosette-negative populations that were then stimulated by PWM and IgG measured.

histamine. Moreover, Con A-induced suppressor activity was also present to the same degree in the unfractionated and E-rosette-positive populations. However, the E-rosette-negative populations preactivated with Con A also induced significant suppression of IgG production, albeit less than that caused by the other populations. The E-rosette-positive population was then treated with monoclonal antibodies directed against T-cell subsets ( 18). A source of complement was provided (guinea pig serum) in an attempt to delete the relevant populations of helper (OKT4+) and suppressor (OKTS+) cells. The remaining treated cells were adjusted to the concentration of the untreated cells and incubated with histamine ( 10e4 M) or Con A (5 rg/ml) for 24 hr, washed, and recombined with autologous E-rosette-negative cells that were then stimulated with PWM. The results of four experiments are summarized in Fig. 4. Histamine-and Con A-pretreated cells both significantly suppressed PWM-induced IgG production when their incubation took place in the presence of buffer or guinea pig serum alone (37 and 75% suppression for histamine and Con A, respectively). Cells treated with OKT4 plus complement were much less able to provide help and therefore IgG synthesis per se was markedly reduced. However, both histamine and Con A were able to proportionally inhibit the amount of IgG made under these conditions compared to cells treated with buffer or guinea pig serum (i.e., the degree of suppression for histamine and Con A was similar to that described above for control cells). In contrast, cells treated with OKT8 plus complement were much less able to suppress IgG following incubation with histamine or Con A. Under these conditions both spontaneous and PWM-induced IgG production were markedly increased. OKT8-treated cells stimulated by histamine or Con A only inhibited IgG production by 9 and 20%, respectively. These

330

LIMA AND ROCKLIN

Cell Treatment

4. Treatment of histamine- and Con A-induced suppressor cells with monoclonal antibodies directed against T-cell subsets. T-Enriched cells were incubated in buffer alone, buffer + complement, OKT4 antiserum + complement, or OKTS antiserum + complement, washed, and then incubated with or without histamine or Con A for 24 hr. After incubation, the remaining cells (OKT4- or OKTS-) were cocultured with autologous E-rosette-negative cells that were then stimulated by PWM. FIG.

levels of suppression are statistically less (P < 0.05) than that obtained for the control-treated cells. These results indicate that both the histamine- and Con Aresponsive suppressor cells are contained within the OKT8+ population. Futhermore, deletion of the OKT4+ population does not appear to alter suppressor activity to any significant degree. E-Rosette-positive cells were exposed to varying concentrations of irradiation and then stimulated with histamine ( 10e4or 10m5M) or Con A (5 pg/ml) for 24 hr. The latter cells were washed and recombined with autologous E-rosette-negative cells that were then stimulated with PWM. As shown in Fig. 5 (mean of four experiments), histamine-induced suppressor activity was not significantly reduced until 2000 rad was applied to the cells. In contrast, Con A-induced suppression was not significantly reduced until 3000 rad was applied. These results suggest that histamine- and Con A-induced suppressor cells have differing sensitivities to irradiation. In related experiments, in order to determine whether B cells were responding to histamine directly, E-rosette-positive cells were radiated with 3000 rad and recombined with E-rosette-negative cells. The latter were stimulated with PWM and 10e4 M histamine was added. The results of these experiments (Table 1) documented that histamine had no direct effect on IgG production, suggesting that under these experimental conditions B cells did not demonstrate functional histamine receptors. Nature of the Histamine

Receptor

In our previous experiments, histamine-induced suppression of lymphocyte proliferation or lymphokine production was mediated through the Hz receptor (3-7).

HISTAMINE

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331

IgG PRODUCTION

0he

of lrradiotion

(rods)

5. Effect of irradiation on histamine and Con A-induced suppressor cells. T-Enriched cells were exposed to 1000-3000 rad and then incubated with histamine or Con A for 24 hr. and washed and coculture was carried out. Histamine-induced suppressor activity was abolished by 2000 rad, while inhibition of Con A-induced suppression activity required 3000 rad. FIG.

In the present studies we sought to describe the nature of the receptor in the histamine-induced suppression of IgG production. For these experiments, E-rosettepositive cells were cultured for 24 hr in the presence of histamine alone, or histamine plus an H, antagonist (diphenhydramine) or an Hz antagonist (cimetidine), washed, and then cocultured with autologous E-rosette-negative cells. Histamine-induced suppression of IgG production was then measured. T-Enriched cells were also cultured with diphenhydramine or cimetidine alone for their effects on IgG production. Three experiments are shown in Table 2. Histamine ( 10e4M) alone caused a 2332% suppression of IgG production. Diphenhydramine alone induced suppressor activity at levels that were even greater than that for histamine. Cimetidine alone had no effect on IgG synthesis. The combination of histamine plus diphenhydramine

TABLE 1 Lack of Direct Effect of Histamine on IgG Production In vitro IgG production (rig/ml)

Experiment I Histamine 04)

Control

None 1O-4 IO+ 1o-6

4500” 3400 (24)* 3500 (22) 3900 (13)

3000 rad 6000 7000 (-16) 7500 (-25) 5900 (2)

Experiment II Control 1800 1000 (44) 1250 (30) 1300 (27)

3000 rad 2700 2500 (7) 2800 (-4) 2800 (-4)

Note. E-Rosette-positive cells were either irradiated (3000 rad) or not irradiated, and cocultured with E-rosette-negative cells that were stimulated by PWM and to which histamine was added. ’ rig/ml IgG in culture supernatant. bPercentage suppression of IgG production in parentheses.

332

LIMA AND ROCKLIN TABLE 2 Effect of H, and Hz Antagonists on Histamine-Induced Suppression of IgG Production’

Drug ( 10e4M)

Experiment I

Experiment II

None Histamine Diphenhydramine Cimetidine Histamine and diphenhydramine Histamine and cimetidine

2600 2000 (23)b 1700 (40) 2500 (4)

1600 1100 (32) 900 (44) 1500 (6)

2000 1500 (25) 1000 (50) 1700 (15)

1500 (12)

900 (0)

1300 (-30)

1000 (33)d

1400 (18)d

1900 (24)d

Experiment III

’ E-Rosette Positive cells were incubated with histamine, diphenhydramine, cimetidine, or histamine plus the H, or H2 antagonists for 24 hr. washed, and cocultured with autologous B cells that were then stimulated with PWM for 7 days. b Numbers in parentheses indicate percentage suppression of IgG synthesis induced by histamine, diphenhydramine, or cimetidine compared to PWM alone. ’ Percentage suppression of IgG synthesis calculated using PWM + diphenhydramine as the “control” value. d Percentage suppression of IgG synthesis calculated using PWM + cimetidine as the “control” value.

did not result in an additive effect. However, histamine plus cimetidine did not alter the level of histamine-induced suppression. E-Rosette-positive cells were also incubated with an Hi agonist (2-pyridylethylamine) or an H2 agonist (dimaprit) for 24 hr, washed, and cocultured with autologous E-rosette-negative cells that were stimulated with PWM. As shown in Fig. 6, in which four experiments are summarized, both the Hi and H2 agonists significantly suppressed IgG production in a dose-dependent manner. Inhibition occurred in the unfractionated population and to the same degree in the rosettepositive population. E-Rosette-negative cells preincubated with either drug failed to inhibit IgG production. Furthermore, E-rosette-positive cells initially exposed to 3000 rad and then to the drugs also failed to inhibit IgG production. The results with the agonists and antagonists suggest that both the Hi and Hz receptor may be involved in activation of histamine-induced suppressor cells involved with IgG regulation. These results are in contrast with histamine-induced suppression of Tcell functions. DISCUSSION The purpose of the present study was to evaluate the immunoregulatory effects of soluble histamine on in vitro IgG production by human mononuclear cells. We found that histamine, when added to cultures of mononuclear cells stimulated with PWM, inhibited IgG production in a dose-dependent manner. IgG was measured in culture supernatants using a double-antibody radioimmunoassay. Histamine had no significant effect on the spontaneous amounts of IgG produced in culture over a 7-day period. Maximum suppression (55%) was achieved with 10e4M histamine. That the inhibitory effect of histamine on IgG production was not merely a toxic one was suggested by kinetic experiments. Histamine added at varying times after the addition of pokeweed mitogen was only effective in significantly suppressing

HISTAMINE o-o

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2 - Pyridylethylamine (H, agorust) Dlmaprit (lip ogonist 1

O-O

m

MODULATES

UNFRACTIONATED

T

CELLS

E-ROSETTE

8

CELLS

t

Drug Concentration

(Ml

FIG. 6. Ability of H, and H, agonists to cause suppression of IgG. T-enriched cells or T-depleted cells were preincubated with 1O-“ to lo-’ M 2-pyridylethylamine or Dimaprit for 24 hr., washed, and cocultured with PWM-stimulated autologous T-depleted cells. In some experiments, T-enriched cells were exposed to 3000 rad prior to incubation with agonists.

IgG production for the first 48-72 hr. After 72 hr, the addition of histamine did not alter IgG levels. A variety of studies in animals has suggested that there are complex interactions between T cells, B cells, and macrophages for the regulation of antibody production (19-21). A number of in vitro studies have been carried out in humans as well in an attempt to analyze immunoregulation of humoral responses. Polyclonal stimulants such as PWM (22-28) and staphylococcal protein A (28, 29) as well as specific stimuli such as TNP-polyacrylamide (30) and tetanus (3 1) have been employed to measure in vitro plaque-forming cells and immunoglobulin content of culture supernatants. Immunoregulation in these studies has been analyzed using either preincubation or aging of cultures (22), concanavalin A (24, 26-28, 30, 3234), sodium periodate (35), or tetanus antigen (3 1) to activate suppressor cells. Suppressor cell activity has been identified in a number of mononuclear subpopulations and these cells have been partially characterized. For example, the suppressor cell induced by aging or preculturing mononuclear cells in vitro has been identified as being a T cell that requires DNA synthesis before being able to express its activity and is hydrocortisone sensitive (22). Spontaneously generated suppressor cells capable of inhibiting pokeweed-induced plaque-forming cells have been identified as being contained within both the Erosette-positive and E-rosette-negative populations (25). The latter suppressor cells have in common, however, the presence of an Fc receptor, are radiosensitive (2000 rad), and also require DNA synthesis before expressing their activity (22-25). In

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LIMA AND ROCKLIN

addition, a radioresistant adherent cell, presumably a macrophage, has also been described as mediating this type of immunosuppression (25). Furthermore, Con Ainduced suppressor cells capable of inhibiting PWM-induced plaque-forming cells or IgG synthesis in vitro have been characterized as being contained. within the Erosette-positive population, are lymphoblastoid, and are sensitive to mitomycin C treatment (24, 26, 28, 30, 32). A Con A-responsive cell is also present within the E-rosette-negative population having Fc receptors for IgG but lacks receptors for complement (24). Con A-reactive suppressor cells were also identified in a T-celldepleted population which was nonadherent and complement receptor negative (24). Recently, differentiation antigens detected by monoclonal antibodies have characterized suppressor cells as being contained within the OKT8+ subpopulation (36, 37). The cell bearing a histamine receptor having immunoregulatory effects on in vitro IgG synthesis has also been partially characterized in the present study and can be contrasted with the above-reported suppressor cells. Using a coculture technique, we found that histamine-responsive suppressor cells were present within the E-rosette-positive population (Fig. 3). This finding was also true for the Con Aactivated suppressor cell response detected in parallel. In addition, the histamineresponsive cell was radiosensitive in that its activity was abolished by exposing Erosette-positive cells to 2000 rad. The Con A-induced suppressor cell activity was not abolished until 3000 rad had been administered. We cannot determine at this point whether the latter results imply that the histamine- and Con A-responsive suppressor cells are within different subpopulations or, if in the same cell, whether the histamine stimulus is a much weaker signal or affects fewer cells than the Con A stimulus. Of particular interest, irradiated T-enriched cells cocultured with Erosette-negative cells to which histamine was added failed to inhibit IgG production, implying that B cells were not directly responsive to histamine. Moreover, the histamine-responsive cell bears the OKT8+ antigen on its surface, since treatment of T-enriched cells with OKT8 antisera and complement abolished histamine-induced suppressor activity (Fig. 4). Consistent with previous findings, the Con Ainduced suppressor cell was also found to possessthe OKT8 antigen (36, 37). In contrast, treatment of E-rosette-positive cells with OKT4 antisera + complement, while significantly reducing pokeweed-driven IgG production per se, did not alter the amount of suppression of IgG compared to control-treated cells. The significance of the latter finding is discussed below. The mechanism by which suppressor cells regulate antibody production has been analyzed to some extent in a number of studies. It appears that suppressor cells must be present early in culture with B cells that have been stimulated by whatever means. In our experiments, the histamine-induced suppression diminished significantly with time after the addition of pokeweed. One study suggested that Con Aactivated suppressor cells act directly on T cells that are involved with cooperation (34). On the other hand, several studies have indicated that suppressor cell activity is expressed only after interactions between precursor suppressor cells and a suppressor cell inducer (26, 37, 38). Furthermore, Haynes and Fauci (33) have suggested that Con A-activated suppressor cells generate an autoreactive population of cells that mediate an autologous lymphocyte reaction against non-T cells that lead to suppression of B-cell function. In the mouse system, Rich and Pierce have demonstrated that Con A-activated

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T cells secrete a soluble substance (soluble immune response suppressor or SIRS) that does not act directly on T cells but rather affects macrophages in such a way that the latter cells then inhibit B-cell function (39). In man, recent studies suggest that human splenic lymphocytes activated with Con A produce a soluble factor that leads to suppression of plaque-forming cells and immunoglobulin secretion (27). While not directly addressed in the present study, we have recently obtained evidence which suggests that the histamine-induced suppressor factor (HSF) may have some direct effects on T-helper-cell function (40). In the latter experiments, human HSF has suppressed T-helper-cell function generated during a mixed lymphocyte reaction. This has lead to decreased production of IgG, IgM, and IgA plaque-forming cells. Of particular interest in the latter studies, HSF has also decreased the appearance of Ia/DR antigen expression on T cells generated during a mixed lymphocyte reaction. We have also observed that Con A- and histamineinduced suppression of lymphocyte proliferation can be blocked by the presence of indomethacin (41). The latter finding would suggest a SIRS-like effect of histamine- or Con A-induced suppressor cells such that the soluble factor thus generated alters monocyte function in a way that results in increased synthesis of prostaglandins. The latter then may be involved in the final pathway leading to suppression. It is not clear at the present time whether the same mechanism applies to histamine- or Con A-induced suppression of immunoglobulin production. The nature of the histamine receptor present on regulatory cells activated by histamine was evaluated in the present study but the results are somewhat open to interpretation. Using Hi and H2 antagonists, the data could suggest that the H, receptor is primarily involved. For example, we noted that the Hi antagonist (diphenhydramine) on its own was capable of suppressing IgG production to a greater extent than even histamine. When histamine and diphenhydramine were combined in equimolar concentrations, instead of obtaining increased activity due to each drug, we essentially obtained an amount of suppression equal to either drug alone (Table 1). The Hz antagonist (cimetidine) had no effect per se on pokeweed mitogen-induced IgG production and, when combined with histamine in equimolar concentrations, essentially did not change the suppression achieved by histamine. Employing H, (2-pyridylethylamine) and H2 agonists (Dimaprit) with the same coculture technique, we noted that both agonists were capable of suppressing IgG production in a dose-dependent manner (Fig. 6). In addition, the Hi and Hz agonistresponsive cell was present within the E-rosette-positive population and was sensitive to 3000 rad. While the interpretation of these data is still open to question, it certainly implies that the Hi receptor, as well as the Hz receptor, is involved in the regulation of IgG production. The finding that both H, and H2 receptors may be involved in modulating IgG production is of interest and contrasts with previous studies involving the effects of histamine on T-cell function (2-7). In the latter studies the Hz receptor was found to be predominantly involved. In summary, we have shown that the addition of soluble histamine in vitro to cultures of human mononuclear cells stimulated with pokeweed mitogen suppresses IgG production by these cells. The histamine-responsive cell is most likely a T cell that is sensitive to irradiation and may be activated through both H1 and Hz receptors to express its regulatory function. We are currently investigating the mechanism of histamine-induced suppression and the nature of the histamine-responsive regulatory cell.

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ROCKLIN

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