CELLULAR
IMMUNOLOGY
The Effect
(1979)
46,360-372
of Histamine
I. Demonstration
on Leukocyte
Migration
of a LIF Production
Inhibitor
D. RIGAL, J. C. MONIER, Laboratoire
de Midecine
August
(LIF-PI)
AND G. SOUWEINE
Prhentive, Faculte de MPdecine Alexis Paradin. 69372 Lyon Cedex 2, France Received
Test in Man
Carrel,
Rue Guillaume
IO, 1978
The inhibition by BCG of leukocyte migration was abolished by histamine in human donors with a positive tuberculin skin test. This effect was related to two mechanisms: a direct stimulation of polynuclear cell migration by histamine, and the production of a LIF production-inhibiting soluble factor (LIF-PI) by nylon-adherent T lymphocytes with receptors for histamine. This factor was not dialyzable, had a molecular weight below 50,000, and was absorbed on aggregated human immunoglobulins. Cells which produced this factor behaved as suppressors of the in vitro human delayed hypersensitivity reaction.
INTRODUCTION When histamine binds to blood vessels, bronchae, and intestine smooth muscle type I histamine receptors (HI receptors) it induces inflammatory effects. On the contrary its binding to leukocyte type II histamine receptors (H2 receptors) causes an anti-inflammatory effect. This anti-inflammatory role of histamine on T lymphocytes is now well documented since it has been shown that histamine inhibits T-lymphocyte cytotoxicity on tumoral cells in mice (l), MIF production by guinea pig T cells (2), proliferation of T lymphocytes following stimulation by either antigen or mitogen in the guinea pig (2,3) and the mouse, proliferation of human lymphocytes induced by phytohemagglutinin (PHA) and concanavalin A (Con A), antibody response to thymodependent antigen in the mouse (3), production of interferon (3), and mixed lymphocyte reaction (MLR) in mice (4). In a previous paper by our laboratory (5), it was shown that histamine prevents the specific inhibition of leukocyte migration in man. The present paper gives new information on the effects of histamine and shows that its action on LIF synthesis is mediated by an inhibitory factor produced by T lymphocytes with a receptor for histamine. MATERIALS
AND METHODS
1. Subjects Healthy volunteers from our laboratory, eight females and six males, aged 20 to 35, gave blood for this study. Ten subjects had positive skin tests to 10 units of 360 OOO8-8749/79/100360-13$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
EFFECT
OF HISTAMINE
ON
LEUKOCYTE
MIGRATION
361
TEST
tuberculin (Institut Pasteur); five had negative skin tests to both 10 and 50 units of tuberculin. In each case 40 ml of venous blood was drawn into a glass tube containing 100 U/ml heparin (Liquemin Roche). 2. Leukocyte
Migration
Test (LMT)
The capillary tube technique described by Soborg and Bendixen (6) was used with Gorski’s modification (7). Blood cells were allowed to sediment for 30 min at 37°C. The supematant was then centrifuged at 4OOg, 10 min at 4°C. The cells were washed twice in Hanks’ medium, pH 7.3 (Bio Merieux). The leukocytes were adjusted to 40 X IO6 cells/ml in RPM1 medium (Bio Merieux) containing 10% fetal calf serum (FCS) (Eurobio), 1% glutamine (Bio Merieux), 200 mmol/ml, 100 pi/ml streptomycin, and 100 III/ml penicillin G; the pH was adjusted to 7.3 by adding 5.6% sodium bicarbonate (Bio Merieux). Two aliquots of this cell suspension were preincubated for 30 min at 37°C one with 15 ~1 PBS, pH 7.2 (Bio Merieux), only (control), the other with 7.5 or 15 pg BCG (Institut Pasteur) in 15 ~1 PBS, pH 7.2. The cell suspensions were then drawn into capillary tubes (Hemocap) of 1.1 mm internal diameter. After centrifugation for 10 min at 2OOg, the tubes were cut at the level of the interface between cells and medium. Tubes were fixed with silicone grease (Rhone Poulenc) to the bottom of migration chambers containing 2 ml of the culture medium. Histamine dihydrochloride (Sigma), 20 mg/ml, in RPM1 (BD Merieux) + 20 m&f Hepes (Sigma) was poured into the migration chamber, giving a final concentration of 100 kg/ml of culture medium. Migration chambers, covered by glass slides fixed by silicone grease, were kept for 18 hr at 37°C. Migration areas were traced onto paper with the aid of an epidiascope, and then cut and weighed. We were thus able to determine a migration inhibition index: weight of migration area with BCG weight of migration area without BCG The migration
inhibition
3. Effect of Histamine
index was the mean of quadruplicate on Mononuclear
x 100. determinations.
(MN) Cells
MN cells, 3 x 106, from four donors with positive tuberculin skin tests were incubated for 30 min at 37°C with 100 pg/ml histamine; histamine was dissolved in RPM1 + 1% glutamine, pH 7.3, without FCS. Cells were washed three times in Hanks’ balanced salt solution and subsequently added to 9 x lo6 PN cells from the same donor. The MN + PN cell suspension was then placed in migration tubes with 15 &ml BCG. The control consisted of MN cells from the same donor, which had not been incubated with histamine. 4. Exclusion of Mononuclear
Cells with a Receptorfor
Supematant from blood sedimented Lymphoprep gradient (Nyegaard). After temperature, the MN cells were then procedure. The resulting cell population contained
Histamine
(MNH)
(Fig. la)
for 30 min at 37°C was poured onto a centrifugation for 20 min at 400g at room further purified, again using the same 98% MN cells and 2% polynuclear
cells
362
RIGAL,
MONIER,
AND
SOUWEINE
a 3
Ficoll
isopaque
sitdimentation45mn3X
-HISTA. Sbpematant =-+ ==+ * +HISTA Supernatant
Dialysis 18hnNACI 0.15 N 6hin TClS6
kiiiii5a TC 196 without serum 24h37C5”hC02
‘:.‘.: :::’.v. El-
Bully coat cdl.3 sedimentation 45mn 37C
-
IJ
40.106Leukocyteslml ~)Mupertant HStor HS _ 15 #I PBS without or with BCG lnwbatiin 37C3Omn
FIG.
1. Preparation
of cellular
RPMMo9/oFCS l8h37C
suspensions
and LIF-PI.
(PN). The cell viability (estimated by trypan blue exclusion test) was 97%. The PN cells collected from the bottom of tubes were washed in Hank’s balanced salt solution and stored until further use in culture medium at 4°C (within 4 hr following preparation). Cells with receptors for histamine were recovered from the mononuclear cell population by the rosette technique with red blood cells coated with histamine, according to the unmodified Kedar and Bonavida technique (8). Histamine dihydrochloride (1.4 g), rabbit serum albumin (200 mg), and ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (1.2 g) (ECDI Sigma) were dissolved in 20 ml phosphate buffer (PBS, 0.01 M, pH 7.2, in NaCl, 0.14 M ). After incubation for 1 hr at room temperature, the solution was dialyzed at 4°C against 2000 ml PBS, which
EFFECT
OF HISTAMINE
ON
LEUKOCYTE
MIGRATION
TEST
363
was changed three times within 48 hr. The conjugate histamine/rabbit albumin (HR) was frozen and used within 1 month following its preparation. The HR conjugate was centrifuged for 15 min at 10,OOOg before coupling with sheep red blood cells (SRBC). Twenty-five milliliters of a 50% suspension of SRBC in PBS was added to 2.5 ml HR conjugate and 1 ml PBS containing40 mg ECDI. After 45 to 60 min incubation at room temperature, conditioned SRBC were washed three times in a cold solution of Ca’+, Mg2+-Dulbecco’s PBS containing 1% inactivated FCS and absorbed on SRBC; they were then adjusted to 2.5 x lOa SRBC-HR/ml. Sheep red blood cells coupled to rabbit serum albumin only and normal SRBC served as controls. One milliliter of a 5 x lo6 MN cells/ml suspension was added to 1 ml of SRBC-HR; the tubes were then centrifuged for 7 min at 175g at 4°C and then kept for 30 min at 4°C. The cells were then gently resuspended at 4°C poured onto a gradient of Ficoll-Isopaque, and spun down for 20 min at 4008 at 4°C. The cells recovered at the interface had no histamine receptors (MNH-). Rosette-forming cells (MNH+) were recovered at the bottom of the tubes after SRBC-HR had been hemolyzed by 0.83% NH&l for 6 min, and were subsequently washed twice in Hanks’ balanced salt solution. The two populations, MNH+ and MNH-, were then reconstituted with PN to a final concentration of 40 x lo6 cells/ml at a ratio MN:PN = 2:3. These populations were then tested in LMT with BCG present with or without histamine. 5. LIF-PI
Production
(Fig. Ib)
The technique of Rocklin (9) was used with some modifications. Mononuclear cells (3 x 106), obtained after centrifugation with a Ficoll-Isopaque gradient of peripheral blood diluted in an equal volume of Hanks’ balanced salt solution, were incubated for 24 hr at 37°C in an incubator containing 95% air + 5% carbon dioxide. The cells were poured into 2-cm-diameter petri dishes (Falcon) with the culture medium: TC 199 (BD Merieux) + streptomycin (100 tLglm1) + penicillin G (100 p/ml) + Hepes (25 mmohliter), with various doses of histamine (supernatant histamine+, SH+) or without histamine (supernatant histamine-, SH-). The culture supernatant was dialyzed for 18 hr against PBS, pH 7.2, and for 6 hr against TC 199. Histamine (100 pg/ml) was added to the control supernatant before dialyzing; 400 ~1 of this latter supernatant + 500 ~1 of a suspension of 40 x lo6 leukocytes/ml from subjects with positive tuberculin skin tests and 15 ~1 PBS, pH 7.2, with or without 15 or 30 pg BCG were incubated for 30 min at 37°C. This suspension was then placed in capillary tubes and LMT was performed. This experiment is illustrated in Fig. 2. 6. LIF Production
and Incubation
with LIF-PI
MN cells, 5 x 106, from donors with positive tuberculin skin tests were incubated in 1 ml RPM1 + 10% FCS + 1% glutamine, pH 7.3, with or without 15 pg/ml BCG, and were kept 24 hr at 37°C in an atmosphere containing 5% CO2 and 95% air. The supematant was collected after centrifugation at 1OOOOgfor 30 min at 4°C. One-half milliliter of supematant with LIF was added to either 0.5 ml of supernatant with LIF-PI or 0.5 ml of supematant without LIF-PI and the mixture was assayed in a PN migration test with PN cells from the LIF-producing donors.
fmm
b-Total leucocytea 3 subjects with skin tests-
fmm
c. PN leaocytesfrom 5 subjects withskin tests*
FIG. 2. Effect of histamine (100 &ml) on LMT-total leukocytes (a and b) or PN cells (c)-in the presence of BCG (15 FL&).Results obtained with five donors with positive tuberculin (10 units) skin tests (a and c) and three donors with negative tuberculin skin tests (b). Each experiment was repeated twice in each donor.
a _ Total leuaxyte8 5 subjects with skin tests +
EFFECT
7. Characterization
OF HISTAMINE
ON LEUKOCYTE
of LIF-PI-Producing
MIGRATION
TEST
365
Cells
E rosette formation. Fournier and Bach’s technique (10) was used with two enrichments with E rosette-forming cells (E-RFC). Adherence to glass and nylon. Eight milliliters of washed and sterilized glass or nylon wool was placed in lo-ml plastic syringes. An equilibrium was established with RPM1 medium with 10% FCS; 1 x lo7 cells were poured through the columns. After incubation for 30 min at 37°C nonadherent cells were eluted with 30 ml of medium maintained at 37°C. Adherent cells were recovered after strongly agitating the glass or nylon wool placed in bottles containing 20 ml of culture medium. The cells were then washed twice in Hanks’ balanced salt solution and tested for their ability to produce LIF-PI. 8. Characterization Molecular weight (MW). The supernatant of a 3 x lo6 cell culture was centrifuged at 1OOOg through an ultrafiltration Diaflo membrane (Centrillo, Amicon) which discriminates molecules with MW approaching 50,000. Absorption on immunoglobulins (Zg). Fifty milligrams human Ig (Merieux) was added in PBS, pH 7.2, heated for 15 min at 63”C, homogeneized, and washed three times in PBS, pH 7.2. Centrifugations were performed at 1OOOOg.Supernatants of MN cell cultures with or without histamine were incubated for 30 min at 37°C with aggregated Ig, subsequently centrifuged at lO,OOOg, and finally tested for their LIF-PI activity. 9. Statistics Significance
was evaluated by using the Mann-Whitney
U test.
RESULTS I. The Effect of Histamine
on the Migration
Inhibition
Test
Histamine, 100 pg/ml, significantly (P < 0.01) decreased the inhibition by 15 pg of heat-inactivated BCG of the migration of leukocytes from subjects with a positive tuberculin skin test (Fig. 2a). Theorically this effect could be related to either a direct stimulating action of histamine on PN migration or an indirect effect by inhibition of LIF. 2. The Effect of Histamine
on Leukocyte
Migration
Histamine (100 @g/ml) enhances the migration of leukocytes from donors sensitized and not sensitized to tuberculin when BCG is not added to the cell suspension (Figs. 2a and b). The same effect is observed when purified polynuclear cells are used (Fig. 2~). Hence histamine stimulates polynuclear cells migration in vitro. However, this result does not exclude the possibility of an action of histamine on LIF production. The following experiments were performed to reveal a possible indirect effect of histamine on LIF production.
366
RIGAL,
____- .---_ - ____--.--
MONIER,
---_------
AND
SOUWEINE
___---_-.__-.-.-
EFFECT
OF HISTAMINE
3. The Effect of-a Transient
ON
LEUKOCYTE
MIGRATION
TEST
367
Contact between MN Cells and Histamine
When MN cells from donors with positive tuberculin skin tests are incubated with histamine and are subsequently added to PN cells, BCG no longer inhibit their migration (Fig. 3a). PN cell migration in the absence of BCG was not inhibited if MN cells had previously been incubated with histamine. Therefore histamine previously bound to MN cells has no stimulatory effect on PN cell migration (Fig. 3a). Hence histamine inhibits specific leukocyte migration inhibition by an indirect mechanism mediated by MN cells. 4. The Effects of Modifying the Number of Mononuclear Cells (MN) with Receptors for Histamine (MNH+) on Specific Leukocyte Migration Inhibition Test with or without Histamine (a) Depletion. The number of peripheral blood MN cells with receptors for histamine was estimated in our laboratory, using Kedar and Bonavida’s histamine rosette technique (8), to 21 2 5% (mean r SD). In three donors with positive tuberculin skin tests, the histamine rosette-forming subpopulation was eliminated from the mononuclear cell population (cf. Materials and Methods). Following this treatment the number of MNH+ cells fell below 2%. The reconstitution, with this kind of cell suspension, of a cellular mixture with a ratio of MN cells to PN cells of 1:2, leads to the disappearance of the effect of histamine on the inhibition by BCG of leukocyte migration and to the increase of the specific inhibition of leukocyte migration (Fig. 3b). (b) Enrichment. When most of the MN cells of the mixture were MNH+ cells (90% ? 2 MNH+), specific leukocyte migration inhibition in the presence of BCG and in the absence of histamine was not observed (Fig. 3~). The former of these observations leads one to think that histamine exerts its effects not only by a direct stimulatory action on polynuclear cell migration, but also indirectly through MNH+ cells which alter the production of LIF. 5. Production by MN Cells, under Histamine Inhibiting LIF Production (LIF-PI)
Mediation,
of a Soluble Factor
The dose-response effect of histamine on lymphocytes is shown in Fig. 4. The dose of 200 pg/ml has been shown to be the lowest required to give a maximum effect. The viability of cells treated by histamine in all cases exceeded 80% (trypan blue exclusion test). MN cells from normal donors, incubated for 24 hr with 100 pg/ml histamine, released in the culture supernatant a dialyzable factor. This factor significantly reduced (P < 0.01) the specific inhibition by BCG of the migration of leukocytes from patients with positive tuberculin skin tests (Fig. 5). When added to previously produced LIF, this factor did not prevent its action (Fig. 6). Thus it may be assumed that it inhibits LIF production. 6. Characterization
of LIF-PI-Producing
Cells
Supernatants of mononuclear cells from three donors enriched in SRBC-rosetteforming T cells, cultured in the presence of histamine, contained LIF-PI.
368
RIGAL, MONIER,
AND SOUWEINE
+ 5 60k g .E so‘+!5 e $ 40# 3020lo-
FIG. 4. The dose-response experiments.)
effect of histamine on LIF-PI production.
(Results are the mean of two
Supernatants from MN cell population cultures in which less than 3% of the cells form SRBC rosettes did not contain LIF-PI (Fig. 7). Thus it may be assumed that T cells are producing LIF-PI. Nylon fibers retained LIF-PI-producing cells and nonadherent cells were unable to produce this factor (Fig. 8a).
a
b
Tcl99
a
b
St%)
a
b
SHf+)
5. Effect on LMT of culture medium TC 199 supematants of MN cell cultures treated (SH+) or not (SH-) with histamine, in the presence of 15 JQJ(a) or 30 /~g (b) BCG. Test cells are circulating leukocytes from donors with positive tuberculin skin tests. The data presented are the mean of the results of eight experiments. FIG.
EFFECT OF HISTAMINE
LIF
ON LEUKOCYTE
SHW
SkII-)
LIF+
MIGRATION
SW+)
LIF+
369
TEST
St40
FIG. 6. Effects on PN cells migration of: LIF, SH+, and SH- cell culture supematants and of mixtures of either LIF+ SH+ or LIF+ SH- cell culture supernatants. (The data presented on Figs. 6,8, and 10 are the mean of the results of seven experiments.)
The passage of MN cell population since either adherent or nonadherent 7. LIF-PI
on glass wool had no discriminating cells produced LIF-PI (Fig. Sb).
effect
Characterization
The supernatants LIF-PI produced.
80
were titered in some experiments
8
to quantitate
the amount of
b
70 60 50 j
4o
2 30 'rc 8 *O *
10
Slit-) of MN’2 cultures
without
ERFC
SHW of ERFC cultun~
FIG. 7. Consequences, on histamine-induced LIF-PI production, of depletion (a) or enrichment of MN cells (h) with T lymphocytes (E-RFC). Supematants are assayed in the presence of BCG (15 pg). on leukocytes from donors with positive tuberculin skin tests. (Results obtained from three experiments.)
RIGAL,
MONIER,
AND
a NYLON
SOUWEINE b
I ! I I I I I I I
WOOL
GLASS
WOOL
! ! I I I ! I I 1 ! I 1 i
FIG.
wool
8. LIF-PI (b).
production
assays
by MN
cells adherent
or nonadherent
to nylon
fibers
(a) or glass
-With 100 &ml of histamine present, the supematant diluted l/8 had no effect on the inhibition of migration (Fig. 9). -With 200 pg/ml of histamine present, an effect on the inhibition of migration was observed with dilutions of the supernatant up to l/16, the dilution l/32 having no effect (Fig. 9). The evaluation of the molecular weight of LIF-PI was made by ultrafiltration through a Diaflo membrane. The results indicated that the major part of the LIF-PI has a molecular weight of less than 50,000 (Fig. 1Oa) because most of the activity was recovered from the ultrafiltrated fraction.
FIG.
9. Dilution
effect
on supernatant
generated
with
100 or 200 &ml
of histamine.
EFFECT
OF HISTAMINE
ON
LEUKOCYTE
MIGRATION
TEST
371
b
eo70. 6050‘to-
I I
i I ! I I I I L I
i
i i ultrafiltered
SHH absorbed
on
aggregated lg
FIG. 10. Evaluation of LIF-PI molecular weight by ultrafiltration (Centriflo) (a). LIF-PI absorption on heat-aggregated Ig (b). LIF-PI is assayed on leukocytes from donors with positive tuberculin skin tests, in the presence of BCG (15 pg).
LIF-PI lob).
absorption
on aggregated Ig showed that it is completely
absorbable (Fig.
DISCUSSION We have demonstrated in the present work that histamine impaired specific leukocyte migration inhibition by two mechanisms: a direct stimulatory effect on polynuclear cell migration, and an indirect effect, the induction of the production, by T lymphocytes with histamine receptors, of a factor inhibiting LIF production (LIF-PI). The existence of this latter mechanism was based on several observations. First, mononuclear cells with histamine receptors, inhibited, under histamine activation, the antigen-specific inhibition of leukocyte migration. Hence histamine acts by the mediatory role of mononuclear cells with histamine receptors. Second, LIF-PI is produced by T lymphocytes because it was only detected in supernatants of cultures containing T cells. Thus it can be concluded that mononuclear cells, having histamine receptors necessary to the inhibitory action of histamine, are T lymphocytes. Hence, LIF-PI is produced by T cells, either T lymphocytes with histamine receptors or T lymphocytes without histamine receptors under the influence of T lymphocytes with histamine receptors. Moreover LIF-PI-producing cells adhere to nylon wool; it has been shown by Saxon et al. (11) that lymphocytes with histamine receptors are adherent to nylon fibers.
372
RIGAL,
MONIER,
AND SOUWEINE
As most macrophages are adherent to glass fibers and as cells not adherent to glass fibers still produce LIF-PI, it is probable that macrophages play no role in LIF-PI production. LIF-PI has a molecular weight comprised between 10,000 (not dialyzable) and approximately 50,000 (passes through a Centriflo membrane). However, all the inhibiting activity was not recovered following this last procedure (Fig. 8a). Thus there may exist either more than one inhibiting factor or more or less polymerized forms of the same inhibiting factor. In addition to IBF (Fridman and Goldstein (12)), LIF-PI is absorbed on aggregated immunoglobulins. IBF, however, seems to interfere with humoral response only (in vitro system). In our assay system, LIF-PI production is nonspecific. Rocklin (9) showed that guinea pig lymphocytes, when histamine was present, released a soluble factor inhibiting MIF production and lymphocyte proliferation when antigen is present. This factor was termed the histamine suppressor factor (HSF). It is probably closely related to the LIF-PI that we have described in man. They both probably belong to the group of nonspecific suppressor-soluble factors (Waksmann) (13). Studies are currently under progress to determine more precisely the characteristics of LIF-PI and its possible effects on other immunological phenomena (antibody production, lymphocyte proliferation, etc.). These effects of histamine on MIF and LIF production indicate that anaphylaxis, whose issue is histamine release, might play an important and as yet undetermined regulatory role in cellular hypersensitivity. A further point, as yet to be made clear, is the absence of migration inhibition, when BCG is present, of MNH+ cells from donors with positive tuberculin skin tests, mixed with PN cells. In this case MNH+ cells may either contain nonsensitized cells or be prevented from or unable to produce LIF. ACKNOWLEDGMENTS This work was supported by grants from INSERM
(ATP 7891) and Fact&e Alexis Carrel (Lyon).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Plaut, M., Lichtenstein, L. M., and Henney, C. S., J. Clin. Invest. 55, 856, 1975. Rocklin, R. E., J. Clin. Invesr. 57, 1051, 1976. Weinstein, Y., and Melmon, K. L., Immunol. Commun. 5, 401, 1976. Schwartz, A., Askenase, P. W., and Gershon, R. K., J. Immunol. 118, 159, 1977. Rigal, D., Remy, C., and Monier, J. C., Ann. Immunol. Inst. Pasteur C. 128, 952, 1977. Soborg, M., and Bendixen, G., Acta Med. Stand. 181, 247, 1967. Gorski, A. J., Orlowski, T., Pomorski, Z., Zelechowska, M., and Kwiek, S., CeN. Zmmunol. 8, 162, 1973. Kedar, E., and Bonavida, B., J. Zmmunol. 113, 1544, 1974. Rocklin, R. E., J. Immunol. 118, 5, 1977. Fournier, C., and Bach, J. F., INSERM 57, 105, 1976. Saxon, A., Morledge, V. D., and Bonavida, B., Clin. Exp. Immunol. 28, 394, 1977. Fridman, W. H., and Goldstein, P., Cell. Zmmunol. 11, 442, 1974. Waksmann, B. H., Cell. Immunol. 21, 161, 1976.
IMMUNOLOGY
The Effect
(1979)
46,360-372
of Histamine
I. Demonstration
on Leukocyte
Migration
of a LIF Production
Inhibitor
D. RIGAL, J. C. MONIER, Laboratoire
de Midecine
August
(LIF-PI)
AND G. SOUWEINE
Prhentive, Faculte de MPdecine Alexis Paradin. 69372 Lyon Cedex 2, France Received
Test in Man
Carrel,
Rue Guillaume
IO, 1978
The inhibition by BCG of leukocyte migration was abolished by histamine in human donors with a positive tuberculin skin test. This effect was related to two mechanisms: a direct stimulation of polynuclear cell migration by histamine, and the production of a LIF production-inhibiting soluble factor (LIF-PI) by nylon-adherent T lymphocytes with receptors for histamine. This factor was not dialyzable, had a molecular weight below 50,000, and was absorbed on aggregated human immunoglobulins. Cells which produced this factor behaved as suppressors of the in vitro human delayed hypersensitivity reaction.
INTRODUCTION When histamine binds to blood vessels, bronchae, and intestine smooth muscle type I histamine receptors (HI receptors) it induces inflammatory effects. On the contrary its binding to leukocyte type II histamine receptors (H2 receptors) causes an anti-inflammatory effect. This anti-inflammatory role of histamine on T lymphocytes is now well documented since it has been shown that histamine inhibits T-lymphocyte cytotoxicity on tumoral cells in mice (l), MIF production by guinea pig T cells (2), proliferation of T lymphocytes following stimulation by either antigen or mitogen in the guinea pig (2,3) and the mouse, proliferation of human lymphocytes induced by phytohemagglutinin (PHA) and concanavalin A (Con A), antibody response to thymodependent antigen in the mouse (3), production of interferon (3), and mixed lymphocyte reaction (MLR) in mice (4). In a previous paper by our laboratory (5), it was shown that histamine prevents the specific inhibition of leukocyte migration in man. The present paper gives new information on the effects of histamine and shows that its action on LIF synthesis is mediated by an inhibitory factor produced by T lymphocytes with a receptor for histamine. MATERIALS
AND METHODS
1. Subjects Healthy volunteers from our laboratory, eight females and six males, aged 20 to 35, gave blood for this study. Ten subjects had positive skin tests to 10 units of 360 OOO8-8749/79/100360-13$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
EFFECT
OF HISTAMINE
ON
LEUKOCYTE
MIGRATION
361
TEST
tuberculin (Institut Pasteur); five had negative skin tests to both 10 and 50 units of tuberculin. In each case 40 ml of venous blood was drawn into a glass tube containing 100 U/ml heparin (Liquemin Roche). 2. Leukocyte
Migration
Test (LMT)
The capillary tube technique described by Soborg and Bendixen (6) was used with Gorski’s modification (7). Blood cells were allowed to sediment for 30 min at 37°C. The supematant was then centrifuged at 4OOg, 10 min at 4°C. The cells were washed twice in Hanks’ medium, pH 7.3 (Bio Merieux). The leukocytes were adjusted to 40 X IO6 cells/ml in RPM1 medium (Bio Merieux) containing 10% fetal calf serum (FCS) (Eurobio), 1% glutamine (Bio Merieux), 200 mmol/ml, 100 pi/ml streptomycin, and 100 III/ml penicillin G; the pH was adjusted to 7.3 by adding 5.6% sodium bicarbonate (Bio Merieux). Two aliquots of this cell suspension were preincubated for 30 min at 37°C one with 15 ~1 PBS, pH 7.2 (Bio Merieux), only (control), the other with 7.5 or 15 pg BCG (Institut Pasteur) in 15 ~1 PBS, pH 7.2. The cell suspensions were then drawn into capillary tubes (Hemocap) of 1.1 mm internal diameter. After centrifugation for 10 min at 2OOg, the tubes were cut at the level of the interface between cells and medium. Tubes were fixed with silicone grease (Rhone Poulenc) to the bottom of migration chambers containing 2 ml of the culture medium. Histamine dihydrochloride (Sigma), 20 mg/ml, in RPM1 (BD Merieux) + 20 m&f Hepes (Sigma) was poured into the migration chamber, giving a final concentration of 100 kg/ml of culture medium. Migration chambers, covered by glass slides fixed by silicone grease, were kept for 18 hr at 37°C. Migration areas were traced onto paper with the aid of an epidiascope, and then cut and weighed. We were thus able to determine a migration inhibition index: weight of migration area with BCG weight of migration area without BCG The migration
inhibition
3. Effect of Histamine
index was the mean of quadruplicate on Mononuclear
x 100. determinations.
(MN) Cells
MN cells, 3 x 106, from four donors with positive tuberculin skin tests were incubated for 30 min at 37°C with 100 pg/ml histamine; histamine was dissolved in RPM1 + 1% glutamine, pH 7.3, without FCS. Cells were washed three times in Hanks’ balanced salt solution and subsequently added to 9 x lo6 PN cells from the same donor. The MN + PN cell suspension was then placed in migration tubes with 15 &ml BCG. The control consisted of MN cells from the same donor, which had not been incubated with histamine. 4. Exclusion of Mononuclear
Cells with a Receptorfor
Supematant from blood sedimented Lymphoprep gradient (Nyegaard). After temperature, the MN cells were then procedure. The resulting cell population contained
Histamine
(MNH)
(Fig. la)
for 30 min at 37°C was poured onto a centrifugation for 20 min at 400g at room further purified, again using the same 98% MN cells and 2% polynuclear
cells
362
RIGAL,
MONIER,
AND
SOUWEINE
a 3
Ficoll
isopaque
sitdimentation45mn3X
-HISTA. Sbpematant =-+ ==+ * +HISTA Supernatant
Dialysis 18hnNACI 0.15 N 6hin TClS6
kiiiii5a TC 196 without serum 24h37C5”hC02
‘:.‘.: :::’.v. El-
Bully coat cdl.3 sedimentation 45mn 37C
-
IJ
40.106Leukocyteslml ~)Mupertant HStor HS _ 15 #I PBS without or with BCG lnwbatiin 37C3Omn
FIG.
1. Preparation
of cellular
RPMMo9/oFCS l8h37C
suspensions
and LIF-PI.
(PN). The cell viability (estimated by trypan blue exclusion test) was 97%. The PN cells collected from the bottom of tubes were washed in Hank’s balanced salt solution and stored until further use in culture medium at 4°C (within 4 hr following preparation). Cells with receptors for histamine were recovered from the mononuclear cell population by the rosette technique with red blood cells coated with histamine, according to the unmodified Kedar and Bonavida technique (8). Histamine dihydrochloride (1.4 g), rabbit serum albumin (200 mg), and ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (1.2 g) (ECDI Sigma) were dissolved in 20 ml phosphate buffer (PBS, 0.01 M, pH 7.2, in NaCl, 0.14 M ). After incubation for 1 hr at room temperature, the solution was dialyzed at 4°C against 2000 ml PBS, which
EFFECT
OF HISTAMINE
ON
LEUKOCYTE
MIGRATION
TEST
363
was changed three times within 48 hr. The conjugate histamine/rabbit albumin (HR) was frozen and used within 1 month following its preparation. The HR conjugate was centrifuged for 15 min at 10,OOOg before coupling with sheep red blood cells (SRBC). Twenty-five milliliters of a 50% suspension of SRBC in PBS was added to 2.5 ml HR conjugate and 1 ml PBS containing40 mg ECDI. After 45 to 60 min incubation at room temperature, conditioned SRBC were washed three times in a cold solution of Ca’+, Mg2+-Dulbecco’s PBS containing 1% inactivated FCS and absorbed on SRBC; they were then adjusted to 2.5 x lOa SRBC-HR/ml. Sheep red blood cells coupled to rabbit serum albumin only and normal SRBC served as controls. One milliliter of a 5 x lo6 MN cells/ml suspension was added to 1 ml of SRBC-HR; the tubes were then centrifuged for 7 min at 175g at 4°C and then kept for 30 min at 4°C. The cells were then gently resuspended at 4°C poured onto a gradient of Ficoll-Isopaque, and spun down for 20 min at 4008 at 4°C. The cells recovered at the interface had no histamine receptors (MNH-). Rosette-forming cells (MNH+) were recovered at the bottom of the tubes after SRBC-HR had been hemolyzed by 0.83% NH&l for 6 min, and were subsequently washed twice in Hanks’ balanced salt solution. The two populations, MNH+ and MNH-, were then reconstituted with PN to a final concentration of 40 x lo6 cells/ml at a ratio MN:PN = 2:3. These populations were then tested in LMT with BCG present with or without histamine. 5. LIF-PI
Production
(Fig. Ib)
The technique of Rocklin (9) was used with some modifications. Mononuclear cells (3 x 106), obtained after centrifugation with a Ficoll-Isopaque gradient of peripheral blood diluted in an equal volume of Hanks’ balanced salt solution, were incubated for 24 hr at 37°C in an incubator containing 95% air + 5% carbon dioxide. The cells were poured into 2-cm-diameter petri dishes (Falcon) with the culture medium: TC 199 (BD Merieux) + streptomycin (100 tLglm1) + penicillin G (100 p/ml) + Hepes (25 mmohliter), with various doses of histamine (supernatant histamine+, SH+) or without histamine (supernatant histamine-, SH-). The culture supernatant was dialyzed for 18 hr against PBS, pH 7.2, and for 6 hr against TC 199. Histamine (100 pg/ml) was added to the control supernatant before dialyzing; 400 ~1 of this latter supernatant + 500 ~1 of a suspension of 40 x lo6 leukocytes/ml from subjects with positive tuberculin skin tests and 15 ~1 PBS, pH 7.2, with or without 15 or 30 pg BCG were incubated for 30 min at 37°C. This suspension was then placed in capillary tubes and LMT was performed. This experiment is illustrated in Fig. 2. 6. LIF Production
and Incubation
with LIF-PI
MN cells, 5 x 106, from donors with positive tuberculin skin tests were incubated in 1 ml RPM1 + 10% FCS + 1% glutamine, pH 7.3, with or without 15 pg/ml BCG, and were kept 24 hr at 37°C in an atmosphere containing 5% CO2 and 95% air. The supematant was collected after centrifugation at 1OOOOgfor 30 min at 4°C. One-half milliliter of supematant with LIF was added to either 0.5 ml of supernatant with LIF-PI or 0.5 ml of supematant without LIF-PI and the mixture was assayed in a PN migration test with PN cells from the LIF-producing donors.
fmm
b-Total leucocytea 3 subjects with skin tests-
fmm
c. PN leaocytesfrom 5 subjects withskin tests*
FIG. 2. Effect of histamine (100 &ml) on LMT-total leukocytes (a and b) or PN cells (c)-in the presence of BCG (15 FL&).Results obtained with five donors with positive tuberculin (10 units) skin tests (a and c) and three donors with negative tuberculin skin tests (b). Each experiment was repeated twice in each donor.
a _ Total leuaxyte8 5 subjects with skin tests +
EFFECT
7. Characterization
OF HISTAMINE
ON LEUKOCYTE
of LIF-PI-Producing
MIGRATION
TEST
365
Cells
E rosette formation. Fournier and Bach’s technique (10) was used with two enrichments with E rosette-forming cells (E-RFC). Adherence to glass and nylon. Eight milliliters of washed and sterilized glass or nylon wool was placed in lo-ml plastic syringes. An equilibrium was established with RPM1 medium with 10% FCS; 1 x lo7 cells were poured through the columns. After incubation for 30 min at 37°C nonadherent cells were eluted with 30 ml of medium maintained at 37°C. Adherent cells were recovered after strongly agitating the glass or nylon wool placed in bottles containing 20 ml of culture medium. The cells were then washed twice in Hanks’ balanced salt solution and tested for their ability to produce LIF-PI. 8. Characterization Molecular weight (MW). The supernatant of a 3 x lo6 cell culture was centrifuged at 1OOOg through an ultrafiltration Diaflo membrane (Centrillo, Amicon) which discriminates molecules with MW approaching 50,000. Absorption on immunoglobulins (Zg). Fifty milligrams human Ig (Merieux) was added in PBS, pH 7.2, heated for 15 min at 63”C, homogeneized, and washed three times in PBS, pH 7.2. Centrifugations were performed at 1OOOOg.Supernatants of MN cell cultures with or without histamine were incubated for 30 min at 37°C with aggregated Ig, subsequently centrifuged at lO,OOOg, and finally tested for their LIF-PI activity. 9. Statistics Significance
was evaluated by using the Mann-Whitney
U test.
RESULTS I. The Effect of Histamine
on the Migration
Inhibition
Test
Histamine, 100 pg/ml, significantly (P < 0.01) decreased the inhibition by 15 pg of heat-inactivated BCG of the migration of leukocytes from subjects with a positive tuberculin skin test (Fig. 2a). Theorically this effect could be related to either a direct stimulating action of histamine on PN migration or an indirect effect by inhibition of LIF. 2. The Effect of Histamine
on Leukocyte
Migration
Histamine (100 @g/ml) enhances the migration of leukocytes from donors sensitized and not sensitized to tuberculin when BCG is not added to the cell suspension (Figs. 2a and b). The same effect is observed when purified polynuclear cells are used (Fig. 2~). Hence histamine stimulates polynuclear cells migration in vitro. However, this result does not exclude the possibility of an action of histamine on LIF production. The following experiments were performed to reveal a possible indirect effect of histamine on LIF production.
366
RIGAL,
____- .---_ - ____--.--
MONIER,
---_------
AND
SOUWEINE
___---_-.__-.-.-
EFFECT
OF HISTAMINE
3. The Effect of-a Transient
ON
LEUKOCYTE
MIGRATION
TEST
367
Contact between MN Cells and Histamine
When MN cells from donors with positive tuberculin skin tests are incubated with histamine and are subsequently added to PN cells, BCG no longer inhibit their migration (Fig. 3a). PN cell migration in the absence of BCG was not inhibited if MN cells had previously been incubated with histamine. Therefore histamine previously bound to MN cells has no stimulatory effect on PN cell migration (Fig. 3a). Hence histamine inhibits specific leukocyte migration inhibition by an indirect mechanism mediated by MN cells. 4. The Effects of Modifying the Number of Mononuclear Cells (MN) with Receptors for Histamine (MNH+) on Specific Leukocyte Migration Inhibition Test with or without Histamine (a) Depletion. The number of peripheral blood MN cells with receptors for histamine was estimated in our laboratory, using Kedar and Bonavida’s histamine rosette technique (8), to 21 2 5% (mean r SD). In three donors with positive tuberculin skin tests, the histamine rosette-forming subpopulation was eliminated from the mononuclear cell population (cf. Materials and Methods). Following this treatment the number of MNH+ cells fell below 2%. The reconstitution, with this kind of cell suspension, of a cellular mixture with a ratio of MN cells to PN cells of 1:2, leads to the disappearance of the effect of histamine on the inhibition by BCG of leukocyte migration and to the increase of the specific inhibition of leukocyte migration (Fig. 3b). (b) Enrichment. When most of the MN cells of the mixture were MNH+ cells (90% ? 2 MNH+), specific leukocyte migration inhibition in the presence of BCG and in the absence of histamine was not observed (Fig. 3~). The former of these observations leads one to think that histamine exerts its effects not only by a direct stimulatory action on polynuclear cell migration, but also indirectly through MNH+ cells which alter the production of LIF. 5. Production by MN Cells, under Histamine Inhibiting LIF Production (LIF-PI)
Mediation,
of a Soluble Factor
The dose-response effect of histamine on lymphocytes is shown in Fig. 4. The dose of 200 pg/ml has been shown to be the lowest required to give a maximum effect. The viability of cells treated by histamine in all cases exceeded 80% (trypan blue exclusion test). MN cells from normal donors, incubated for 24 hr with 100 pg/ml histamine, released in the culture supernatant a dialyzable factor. This factor significantly reduced (P < 0.01) the specific inhibition by BCG of the migration of leukocytes from patients with positive tuberculin skin tests (Fig. 5). When added to previously produced LIF, this factor did not prevent its action (Fig. 6). Thus it may be assumed that it inhibits LIF production. 6. Characterization
of LIF-PI-Producing
Cells
Supernatants of mononuclear cells from three donors enriched in SRBC-rosetteforming T cells, cultured in the presence of histamine, contained LIF-PI.
368
RIGAL, MONIER,
AND SOUWEINE
+ 5 60k g .E so‘+!5 e $ 40# 3020lo-
FIG. 4. The dose-response experiments.)
effect of histamine on LIF-PI production.
(Results are the mean of two
Supernatants from MN cell population cultures in which less than 3% of the cells form SRBC rosettes did not contain LIF-PI (Fig. 7). Thus it may be assumed that T cells are producing LIF-PI. Nylon fibers retained LIF-PI-producing cells and nonadherent cells were unable to produce this factor (Fig. 8a).
a
b
Tcl99
a
b
St%)
a
b
SHf+)
5. Effect on LMT of culture medium TC 199 supematants of MN cell cultures treated (SH+) or not (SH-) with histamine, in the presence of 15 JQJ(a) or 30 /~g (b) BCG. Test cells are circulating leukocytes from donors with positive tuberculin skin tests. The data presented are the mean of the results of eight experiments. FIG.
EFFECT OF HISTAMINE
LIF
ON LEUKOCYTE
SHW
SkII-)
LIF+
MIGRATION
SW+)
LIF+
369
TEST
St40
FIG. 6. Effects on PN cells migration of: LIF, SH+, and SH- cell culture supematants and of mixtures of either LIF+ SH+ or LIF+ SH- cell culture supernatants. (The data presented on Figs. 6,8, and 10 are the mean of the results of seven experiments.)
The passage of MN cell population since either adherent or nonadherent 7. LIF-PI
on glass wool had no discriminating cells produced LIF-PI (Fig. Sb).
effect
Characterization
The supernatants LIF-PI produced.
80
were titered in some experiments
8
to quantitate
the amount of
b
70 60 50 j
4o
2 30 'rc 8 *O *
10
Slit-) of MN’2 cultures
without
ERFC
SHW of ERFC cultun~
FIG. 7. Consequences, on histamine-induced LIF-PI production, of depletion (a) or enrichment of MN cells (h) with T lymphocytes (E-RFC). Supematants are assayed in the presence of BCG (15 pg). on leukocytes from donors with positive tuberculin skin tests. (Results obtained from three experiments.)
RIGAL,
MONIER,
AND
a NYLON
SOUWEINE b
I ! I I I I I I I
WOOL
GLASS
WOOL
! ! I I I ! I I 1 ! I 1 i
FIG.
wool
8. LIF-PI (b).
production
assays
by MN
cells adherent
or nonadherent
to nylon
fibers
(a) or glass
-With 100 &ml of histamine present, the supematant diluted l/8 had no effect on the inhibition of migration (Fig. 9). -With 200 pg/ml of histamine present, an effect on the inhibition of migration was observed with dilutions of the supernatant up to l/16, the dilution l/32 having no effect (Fig. 9). The evaluation of the molecular weight of LIF-PI was made by ultrafiltration through a Diaflo membrane. The results indicated that the major part of the LIF-PI has a molecular weight of less than 50,000 (Fig. 1Oa) because most of the activity was recovered from the ultrafiltrated fraction.
FIG.
9. Dilution
effect
on supernatant
generated
with
100 or 200 &ml
of histamine.
EFFECT
OF HISTAMINE
ON
LEUKOCYTE
MIGRATION
TEST
371
b
eo70. 6050‘to-
I I
i I ! I I I I L I
i
i i ultrafiltered
SHH absorbed
on
aggregated lg
FIG. 10. Evaluation of LIF-PI molecular weight by ultrafiltration (Centriflo) (a). LIF-PI absorption on heat-aggregated Ig (b). LIF-PI is assayed on leukocytes from donors with positive tuberculin skin tests, in the presence of BCG (15 pg).
LIF-PI lob).
absorption
on aggregated Ig showed that it is completely
absorbable (Fig.
DISCUSSION We have demonstrated in the present work that histamine impaired specific leukocyte migration inhibition by two mechanisms: a direct stimulatory effect on polynuclear cell migration, and an indirect effect, the induction of the production, by T lymphocytes with histamine receptors, of a factor inhibiting LIF production (LIF-PI). The existence of this latter mechanism was based on several observations. First, mononuclear cells with histamine receptors, inhibited, under histamine activation, the antigen-specific inhibition of leukocyte migration. Hence histamine acts by the mediatory role of mononuclear cells with histamine receptors. Second, LIF-PI is produced by T lymphocytes because it was only detected in supernatants of cultures containing T cells. Thus it can be concluded that mononuclear cells, having histamine receptors necessary to the inhibitory action of histamine, are T lymphocytes. Hence, LIF-PI is produced by T cells, either T lymphocytes with histamine receptors or T lymphocytes without histamine receptors under the influence of T lymphocytes with histamine receptors. Moreover LIF-PI-producing cells adhere to nylon wool; it has been shown by Saxon et al. (11) that lymphocytes with histamine receptors are adherent to nylon fibers.
372
RIGAL,
MONIER,
AND SOUWEINE
As most macrophages are adherent to glass fibers and as cells not adherent to glass fibers still produce LIF-PI, it is probable that macrophages play no role in LIF-PI production. LIF-PI has a molecular weight comprised between 10,000 (not dialyzable) and approximately 50,000 (passes through a Centriflo membrane). However, all the inhibiting activity was not recovered following this last procedure (Fig. 8a). Thus there may exist either more than one inhibiting factor or more or less polymerized forms of the same inhibiting factor. In addition to IBF (Fridman and Goldstein (12)), LIF-PI is absorbed on aggregated immunoglobulins. IBF, however, seems to interfere with humoral response only (in vitro system). In our assay system, LIF-PI production is nonspecific. Rocklin (9) showed that guinea pig lymphocytes, when histamine was present, released a soluble factor inhibiting MIF production and lymphocyte proliferation when antigen is present. This factor was termed the histamine suppressor factor (HSF). It is probably closely related to the LIF-PI that we have described in man. They both probably belong to the group of nonspecific suppressor-soluble factors (Waksmann) (13). Studies are currently under progress to determine more precisely the characteristics of LIF-PI and its possible effects on other immunological phenomena (antibody production, lymphocyte proliferation, etc.). These effects of histamine on MIF and LIF production indicate that anaphylaxis, whose issue is histamine release, might play an important and as yet undetermined regulatory role in cellular hypersensitivity. A further point, as yet to be made clear, is the absence of migration inhibition, when BCG is present, of MNH+ cells from donors with positive tuberculin skin tests, mixed with PN cells. In this case MNH+ cells may either contain nonsensitized cells or be prevented from or unable to produce LIF. ACKNOWLEDGMENTS This work was supported by grants from INSERM
(ATP 7891) and Fact&e Alexis Carrel (Lyon).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Plaut, M., Lichtenstein, L. M., and Henney, C. S., J. Clin. Invest. 55, 856, 1975. Rocklin, R. E., J. Clin. Invesr. 57, 1051, 1976. Weinstein, Y., and Melmon, K. L., Immunol. Commun. 5, 401, 1976. Schwartz, A., Askenase, P. W., and Gershon, R. K., J. Immunol. 118, 159, 1977. Rigal, D., Remy, C., and Monier, J. C., Ann. Immunol. Inst. Pasteur C. 128, 952, 1977. Soborg, M., and Bendixen, G., Acta Med. Stand. 181, 247, 1967. Gorski, A. J., Orlowski, T., Pomorski, Z., Zelechowska, M., and Kwiek, S., CeN. Zmmunol. 8, 162, 1973. Kedar, E., and Bonavida, B., J. Zmmunol. 113, 1544, 1974. Rocklin, R. E., J. Immunol. 118, 5, 1977. Fournier, C., and Bach, J. F., INSERM 57, 105, 1976. Saxon, A., Morledge, V. D., and Bonavida, B., Clin. Exp. Immunol. 28, 394, 1977. Fridman, W. H., and Goldstein, P., Cell. Zmmunol. 11, 442, 1974. Waksmann, B. H., Cell. Immunol. 21, 161, 1976.
