Histamine suppression of human lymphocyte responses to mitogens

CELLULAR

IMMUNOLOGY

Histamine

36, 2%36 (1978)

Suppression of Human Lymphocyte

Responses to Mitogens’

S. RAY WANG AND BURTON ZWEIMAN Allergy

and Imm~~zology Section, Department of Medicine, University of Pennsylvania School of Medicine, and the Veteralz’s Administratio.n Hospital, Philadelphia, Pennsylvania 19104 Received July M,l977

Exogenously added histamine in non-cytotoxic concentrations (10-n-10-a M) suppresses in vitro proliferation of lymphocytes induced by PHA or Concanavalin A. This suppressive effect was observed when histamine was present for as short as ‘, hr in the beginning of the culture. Histamine, in concentrations as high as IO-’ M, did not cause increased release of isotope from Yr-labeled lymphocytes following 4 hr of incubation. The histamine Hz receptor antagonist, metiamide, but not the H1 receptor antagonists diphenhydramine or chlorpheniramine, blocked the histamine suppressive effect. Some of the biological implications of these findings are discussed.

INTRODUCTION Recent studies have suggested that histamine may exert multiple effects on immune reactivity in addition to the traditional association of this agent with immediate hypersensitivity reactions. Besides the inhibition of IgE mediated release of

histamine from mast cells and basophils (1 ), histamine may suppress chemotaxis of eosinophils (2) and basophils (3). Also, experimental animal studies have shown histamine-induced inhibition of the expression of delayed hypersensitivity (4) and of antigen-stimulated reactivities such as lymphocytotoxicity (5) and MIF production (4). There is less published information about histamine effects on human lymphocyte reactivity. Recently, Ballet and Merler (6) reported in limited studies that in vitro addition of histamine, in a single, relatively high ( 1O-3M) concentration suppressed mitogen and antigen-induced lymphocyte proliferation. However, possible toxic effects of this histamine dose were not ruled out, nor were the effects of other concentrations and duration of incubation investigated. We report here dose and duration response effects of histamine on proliferative responses of lymphocytes from atopic and on-atopic human subjects. The effects of Hz-receptor blocking agents are also evaluated. MATERIALS

AND

METHODS

1. Sub ject.s Nineteen human volunteers were screened for atopy by careful history and skin testing with a panel of environmental antigens commonly eliciting allergic reactions. 1 This research was supported in part by NIH Grant No. lT32-AI R01 AI 14332, and a research grant from the Veterans Administration. 28 0008-8749/78/0361-0028$02.00/O Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

07031, NIH

Grant No.

HISTAMINE

SUPPRESSION

OF LYMPHOCYTE

PROLIFERATION

29

Nine subjects were classified as atopic (6 with allergic rhinitis, 2 with extrinsic asthma, 1 with both) ; 10 subjects were non-atopic. Subjects who could not be classified readily were not used in this study. 2. Lymphocyte Separation and Culture Technique Lymphocytes were separated and cultured by a modification of a method previously reported from our laboratory (7). Ten ml of heparinized (20 u/ml) blood was layered over a 5 ml gradient of Ficoll (Pharmacia, Piscataway, N.J.) and Hypaque (Winthrop Labs., New York, N.Y.), specific gravity 1.077, and centrifuged at 900 g for 20 min at room temperature. The cell layer at the interface was removed, washed twice in sterile Hank’s balanced salt solution (HBSS) and suspended at 2 X lo6 lymphocytes per ml of 2 x culture media containing RPMI-1640 (Microbiological Associates) supplemented with L-glutamine (30 mn/r ) , penicillin (200 u/ml), streptomycin (200 pg/ml) (all from Grand Island Biological Co., Grand Island, N.Y.) and 20% (v/v) fetal calf serum (FCS), (Microbiological Associates, Bethesda, Md.). Cell suspensions thus prepared were SS-90% lymphocytes, l&15% monocytes, and occasional granulocytes. Viability was > 95% by trypan blue exclusion. 3. Histamine

Effect on Lymphocyte Proliferative

Response to Mitogens

Lymphocytes, 2 x lO”/ml, were mixed with equal volume of phytohemagglutin (PHA), 4 ~g/ml, (Lot HA-16, Burroughs Wellcome Research, Triangle Park, N.C.). One tenth milliliter of this mixture (containing 2 pg/ml PHA and 1 x lo5 cells) was dispensed into replicate wells of a sterile microtiter plate (Model ISMCV-96-Tc, Limbro Products, New Haven, Corm.). To replicate wells was added 0.1 ml RPMI-1640 (control) or varying concentrations of histamine dihydrochloride (Calbiochem, San Diego, Calif.) in RPMI-1640. The cells were incubated at 37°C in 5% CO/air for 3 days, pulse labeled for 4 hr terminally with [3H]thymidine (specific activity 6.7 Ci/mM, New England Nuclear Co., Boston, Mass.) 0.5 ,.&I/ well and harvested with an automatic cell harvester (Model M24V, Brandall, Rockville, Md.). Isotope uptake was estimated by standard toluene based scintillation counting techniques and was expressed as the mean cpm of each set of 46 replicates. In a similar way, the effects of added histamine and control solution on concanavalin A (Con A) (final concentration 3 pg/ml) stimulated lymphocyte proliferation was assayed. 4. Time Course of Histamine Eflects on Lymphocyte Proliferation In selected experiments, ceils, PHA and histamine were added into each well at the onset of culture as noted above. At designated time intervals, separate plates containing replicate sets of cultures with varying concentrations of histamine were centrifuged at 1000 rpm ; (International Centrifuge, Model PR- J) , the supernatant was removed, the cell pellet was washed once with HBSS and then resuspended in 0.2 ml of fresh culture media without added mitogen or histamine. The plates were then incubated at 37°C for an additional period for a total of 72 hr, with [3H]thymidine added for the final 4 hr of incubation.

30

WANG

,o&d-;

AND

ZWEIMAN

------_ +

SO.80. -

+ PHA

2 0

5 z

70.-

% 8 z

60.-

..

f

2 8 8 100

------

-_--_-_

__-----

--__-_--_-____

so 80 2

/

-PHA ::>:g

0

lO-‘O

I 6’

HLSTAMJNE

Id’

IOCONCENTRATION

M?lorl

FIG. 1. Histamine effects on lymphocyte proliferation in the presence and absence of PHA. Upper panel shows the effects of histamine in varying concentrations on PHA-incubated cultures. Lower panel shows the effects of histamine on cultures not containing PHA.

+ *P

< 0.01.

mean cpm in the presence of histamine

mean cpm in the absence of histamine

x

100 f

SEM.

** P < 0.001.

5. Cytotoxicity

Studies

A possible toxic effect of the added histamine on the cultured lymphocytes was investigated in 2 ways. a. In selected representative cultures, the exclusion of added 1% trypan blue was determined in PHA-cultured lymphocytes with and without added histamine. Cell preparations were inspected for the % of stained cells 5 minutes after addition of the trypan blue. b. Xr release studies were performed by a modification of a method previously described from our laboratory (8). In brief, 2 x lo6 lymphocytes were incubated with Nars1Cr04 (20 &i/ml) .at 37°C for 1 hr in a shaking water bath, then washed twice and resuspended at a concentration of 1 X 106/ml in culture medium. Half ml of the cell suspension and equal volume of varying concentrations of histamine solutions were mixed in a test tube to obtain the same concentrations used in the cell culture. Following incubation for 4 hr at 37”C, the tubes were centrifuged. The supernate of each tube was transierred to a counting vial to which was added the respective supernatant after each cell button was washed with fresh medium and centrifuged (total called a). The cell button was then resuspended in fresh medium, and the cells disrupted by incubation in boiIing water for 10 min. Following centrifugation at 2000 rpm the supernatant of the disrupted cells (called b) was

HISTAMINE

SUPPRESSION

OF LYMPHOCYTE

31

PROLIFERATION

TABLE 1 PHA-Induced Lymphocyte Proliferation in the Presenceof Different Concentrations of Histamine

-

Histamine concentration (M)

Atopic (n = 9) Non-atopic (n = 10)

10-d

10-s

10-l”

10-s

10-e

10-S

97 f 5” 105 f 3

96 & 5 106 f 3

92 f: 4

86 f 4

82 f 4” 70 f 4c

95 f

88zk4<

81 ~4~

3

67f3”

cpm in the presenceof histamine x 100 i SEM. rpm in the absenceof histamine bP < 0.05. “P < 0.01. d P < 0.001

* Mean of

removed for counting. The ratio of a/(a i b) was used as measure of cytotoxicity in comparison with that isotope released from disrupted cells. 6. Histamine-Receptor

Antagonist

Studies

To help determine whether any observed effects of histamine on stimulated lymphocytes were mediated through the HI or Hz receptors (9), experiments were set up with mixtures of appropriate concentrations of histamine with varying concentrations of either the Hz receptor antagonist metiamide (lot. No. 92058, kindly supplied by Smith, Kline and French Laboratories, Philadelphia, Pa.) or the HI receptor antagonists chlorpheniramine (kindly supplied by Schering Labs., Union, N.J.) or diphenhydramine (Parke-Davis and Co., Detroit, Mich.). One hundred microliter aliquots of these mixtures were added to replicate culture wells each containing 1 X 109 lymphocytes and 0.1 ml of 2 microgram/ml PHA. Following incubation at 37°C in 5% Cop/air f or 3.5 hr, the cells were centrifuged, washed once with HBSS and resuspended in fresh medium without added stimulants, histamine or antihistamine agents. Following 69 additional hours of culture with a terminal 4 hr[3H] thymidine labeling period, the cells were harvested and isotope uptake determined as described above. RESULTS 1. EfJects of Ya+;,g

Concentrations

of Histanhe

on Lymph~ocyte Prolifcrntion

Because of considerable variation in the extent of [3H] thymidine incorporation by the lymphocytes of different subjects, the effect of added histamine on this response was expressed as a percentage of that isotope uptake seen in replicate cultures of the same cells without added histamine. The mean percentage isotope uptake into PHA-incubated cells of all subjects at each concentration of added histamine is shown in Fig. 1. At very low (IO-lo M) histamine concentration, no effect was observed. Starting at histamine concentrations as low as 1O-5 N, significant suppression (P < 0.01) was observed; this suppression was progressively more prominent in cultures containing increasing histamine concentration to the highest level ( 1O-3M) tested.

32

WANG

AND

ZWEIMAN

Similar comparisons of histamine effects on [3H] thymidine uptake into nonstimulated lymphocyte showed no consistent pattern (Fig. 1). The considerable variability observed may be due to the relatively low levels of isotope uptake into these unstimulated cells, even in the absence of added histamine. The patterns of histamine-induced suppressive effects were not significantly different when comparing the lymphocytes of atopic and non-atopic subjects. (Table 1). 2. Comparative Effects of Histahae of Culture

Added for Varying Duration

at the Inception

Removal of the supernatant, twice-washing and resuspension of the cells in fresh media effectively removed histamine from the cultures. Analysis of the residual fluid by an enzymatic assay (lo), showed histamine concentrations of 10-r M or less ; these concentrations were already noted (see above) to exert no suppressive effect on isotope incorporation. Results of studies of the suppressive effect on PHA-induced proliferation of histamine present in the culture medium for varying duration after inception of the culture are shown in Table 2. Significant suppression by both histamine lo-* M and histamine 10e3 M was seen in cultures containing histamine for as little as the first 30 min of incubation (P < 0.01 by paired t test for each histamine concentration compared with replicate cultures without histamine). This suppression continued to be significant when histamine was incubated for the varying time periods studied up to 72 hr. For purposes of simplicity, PHA-induced responses for each of the 8 subjects is shown only for 4 hr and 72 hr in Table 2. TABLE Lymphocyte Incubation

Proliferation

with

IXtferent

2

Period of Contact Histamine

period 0

lo-”

with Histamine

and PHA

concentration 10-a M

M l_l_

0.5 hour

72 hours

Qcpm, [“HI thymidine b Paired t test analysis

71,014 126,760 55,142 -

85,000” 135,720 74,218 -

76,807 128,240 61,265 -

12,280 13,803 11,238 27,514

9,074 9,630 9,996 26,232

8,902 8,543 7,219 26,101

94,421 118,149 47,635 41,063 37,657 44,863 24,691 41,973

60,687 99,978 40,004 28,722 24,391 34,778 19,829 33,788

42,482 71,398 39,140 26,429 18,091 28,139 17,163 29,064

incorporation. of isotope incorporation

P < O.Olb

P < 0.01

compared

to culture

without

P < 0.01

P < 0.01

added histamine.

HISTAMINE

SUPPRESSION

OF LYMPHOCYTE

TABLE Lymphocyte

Wr

Expt.

0 lo-& M 10-J M lo+ M

25” 24 20 22

__

-

” cpm in the culture supernatant releasable cpm

33

3

Release in the Presence of Different Histamine concentration

PROLIFERATION

1

Concentrations Expt.

20 28 23 21

of Histamine

2

-

x 100.

These results were analyzed to determine whether histamine present for very short period (e.g., the first 3 hr of culture) was as suppressive as when histamine was present throughout the duration of culture. The relative degrees of suppression by histamine 1O-3 M present for 3 hr (as compared with non-histamine containing cultures, t = 3.30, P < 0.01) was similar with histamine present for 48 hr (t = 3.55, P < 0.01) or 72 hr (t = 3.67, # < 0.01). Similar results were also obtained at histamine concentration of 10m4M. 3. Cytotoxicify

Studies

A possible cytotoxic effect of histamine on the proliferating lymphocytes was first checked by trypan blue exclusion studies. Dye was excluded by 7090% of PHAcultured lymphocytes with no significant decrease at any concentration of added histamine up to and including 10e3M. “‘Cr release studies were performed in cells of 2 subjects selected at random. Addition of varying concentrations of histamine (to 10m3JJ) for 4 hr led to no increased 51Cr release as compared to replicate cultures without added histamine (Table 3). In these experiments, approximately 70% of cellular 51Cr was released by boiling. 4. Histamine Receptor Antpgonist

Studies

In 3 experiments, varying concentrations of the metiamide, diphenhydramine or chlorpheniramine were added to cultures containing added histamine in varying concentrations as well. Following incubation for 34 hr at 37”C, the cells were washed and resuspended in fresh media as noted above. In the presence of metiamide, the expected histamine ( 1O-3iU) induced suppression was blocked completely in 2 experiments, and partially in the 3rd. (Fig. 2). By contrast, diphenhydramine and chlorpheniramine did not block the effects of lo-’ M or 1W M (Fig. 2) of histamine. Metiamide alone in the cell culture did nbt affect PI-IA-induced proliferation. The lymphocyte proliferation induced by PHA in the presence of 1O-5M metiamide was 101 * 2% (SEM) of the control (i.e., in the absence of metiamide), and in the presence of 10d4M metiamide was 100 + 2% (SEM) of the control. Therefore, the effect of metiamide on lymphocyte proliferation is negligible at the concentration of metiamide up to lo-* M for 3fr hr of incubation.

34

WANG

-

AND

ZWEIMAN

diphen hydramine

CONCENTRATION OF ANTIHISTAMINE

(MafOr)

FIG. 2. Histamine

receptor/antagonist effects on histamine suppression of lymphocyte proliferation. Varying concentrations of metiamide (upper panel), diphenhydramine and chlorpheniramine (lower panel) were incubated with lymphocytes and PHA in the presence of histamine lo-’ M for the initial 3+ hr of culture. (0) Expt. 1, (0) Expt. 2, (A) Expt. 3. Percent Isotope Incorporation (at each concentration of drug)

=

cpm in presence of histamine cpm in absence of histamine

5. Histamine Efects on Con-A Stimulated

x 100.

Cultures

To rule out the possibility that the effects of added histamine were directed specifically against PHA or receptors for this mitogen, similar studies were performed in Con-A stimulated lymphocytes from 4 atopic and 2 non-atopic donors. Patterns of suppression similar to that observed with PHA-stimulated cells were observed (Table 4). DISCUSSION The findings described here show that added histamine in non-cytotoxic concentrations significantly reduce the in vitro DNA synthesis by human lymphocytes TABLE Con-A

Histamine Mean

4

Induced Lymphocyte Proliferation in the Presence of Different Concentrations of Histamine 1O-8

10-S

9

88 z!c 5

73 zt P

cpm in the presence of histamine cpm in the absence of histamine

x loo f

concentration,

(n = 6)

A4

1O-10 96 f

0P < 0.05. b P < 0.01. p Mean of

SEM.

10-4

10-h 65 f

6b

69 f

10-Z 6b

68 f

ga

HISTAMINE

SUPPRESSXON

OF LYMPHOCYTE

PROLIFERATION

35

incubated with the mitogens PHA or Con A. This partial suppression requires the presence of histamine only during the first 4 hr of culture, and is more likely mediated through histamine binding to the Hz receptor rather than the HI receptor on the cells. Some of these findings in human cells are quite similar to that observed by Rocklin (4) in antigen-stimulated guinea pig lymphocytes. He showed that histamine at concentrations of 1O-6 to lo-* M suppressed lymphocyte proliferation. In our study, histamine in concentrations as low as 1O-5 M suppressed PHA-induced lymphocyte DNA synthesis, This was also reported by Artis et al. in abstract form (11). The mechanisms underlying this effect are not well-defined. However, several lines of evidence suggest that an increase in lymphocyte cyclic-AMP may be important. Lichtenstein (12) found that histamine in concentrations of 10m6to lOA4 hf increased lymphocyte cyclic-AMP. Smith et al. (13) showed that cyclic-AMP suppressed PHA-induced human lymphocyte proliferation. It is also of note that Bellet and Merler (6) found that phytomitogens stimulate only those T lymphocytes bearing histamine receptors. Suppression by histamine of lymphocyte-mediated cytotoxicity, another putative T-cell mediated response has also been reported. The lymphocytes suppressed in this reaction were those bearing histamine receptors (6). Plaut rf al. (14) found that H, receptor antagonists blocked not only the suppressive effect of histamine on the cytotoxicity, but also the concomitant histamine-induced increase in lymphocyte cyclic AMP. In our study, histamine suppression of PHA induced lymphocyte proliferation was also blocked by an Hz receptor antagonist. Thus, it is certainly conceivable that addition of histamine to mitogen-incubated human lymphocytes inhibits the activation of T-cells bearing Hz histamine receptors, through increase in intracellular cyclic AMP. The biological implications of this in vitro histamine effect are unclear at present. Our finding that lymphocytes of atopics and non-atopics were affected to similar degree by histamine suggest that the cells of the former are not relatively unresponsive because of increased histamine released in viva. Our preliminary studies indicate that the amount of histamine contained in the cultured leukocytes themselves, if released, would not be sufficient to suppress lymphocyte responsiveness. However, it is certainly conceivable that higher concentrations of histamine could be released locally in vivo following immediate hypersensitivity reactions occurring in sites rich in mast cells and/or basophils. If release of such levels of histamine does occur in Z&JO,inhibition of lymphocyte reactivity could lead to diverse effects on immune responses. It has been shown (4) that histamine given locally in viz~o may depress the expression of delayed hypersensitivity. Therefore, it is possible that histamine released during antigeninduced IgE mediated immediate hypersensitivity reactions in zG0 could reduce the extent of the subsequent delayed hypersensitivity reaction directed against the same antigen. Also, it is conceivable that released histamine could alter the activation of those T-lymphocytes which modulate IgE antibody production in certain tissue sites, possibly leading to self-limitation of that type of immune reactivity. Additional studies will be required to clarify these events.

36

WANG

AND

ZWEIMAN

ACKNOWLEDGMENTS The histamine determinations were kindly performed by Dr. Eliot H. Dunsky and Mrs. Joyce Sabof.

REFERENCES 1. Lichtenstein, L. M., and Gillespie, E., Nature 244, 287, 1973. 2. Clark, R. A. F., Sandier, J. A., Gallin, J. I., and Kaplan, A. P., J. Immunol. 118, 137, 1977. 3. Lett-Brown, M. A., and Leonard, E. J., J. Imnzulzol. 118, 815, 1977. 4. Rocklin, R. E., j. Clifs. Invest. 57, 1051, 1976. 5. Plaut, M., Lichtenstein, L. M., Gillespie, E., and Henney, C. S., J. Zmmuuol. 111, 389, 1973. 6. Ballet, J. J., and Merler, E., Cell. Zmmzcnol.24, 250 ,1976. 7. Levinson, A. I., Lisak, R. P., and Zweiman, B., Cell. Zmmzmol. 14, 321, 1974. 8. Zweiman, B., and Miller, M. F., Int. Arch. Allergy Appl. Immus. 46, 822, 1974. 9. Ash, A. S. F., and Schill, H. O., Br. J. Pharmucol. Chemother. 27,427, 1966. 10. Levy, D. A., and Widra, M., J. Lab. CEin. Med. 81,291, 1973. 11. Artis, W. M., Jones, H. E., and Blazkovec, A. A., Fgd. Proc. 34, 1002, 1975. 12. Lichtenstein, L. M., Znt. Arch. Allergy Appl. Immun. 49, 143, 1975. 13. Smith, J. W., Steiner, A. L., and Parker, C. W., J. C&z. Zplvest. 50, 442, 1971. 14. Plaut, M., Lichtenstein, L. M., and Henney, C. S., J. Clin. Invest. 55, 856, 1975.