Immunology, 1975, 29, 223.

Human Polymorphonuclear Leucocyte Migration Inhibitory Factor EVIDENCE FOR ANTIGEN DEPENDENCY R. H. WEISBART, JUDITH ISAACSON, R. BLUESTONE AND L. S. GOLDBERG Department of Medicine, University of California at Los Angeles School of Medicine, and Medical and Research Services, Veterans Administration Wadsworth Hospital Center, Los Angeles, California, U.S.A.

(Received 29th June 1974; acceptedfor publication 3rd February 1975) Summary. Studies were performed on human polymorphonuclear leucocyte migration inhibitory factor (PMN-MIF) to determine its antigen dependence. PMN-MIF produced by lymphocytes in response to purified protein derivative or coccidioidin was measured in an agarose gel system with buffy coat leucocytes as indicator cells. PMN-MIF activity contained in the lymphocyte supernatants uniformly disappeared when the supernatants were diluted 1:50 with medium; the inhibitory activity was only restored when the diluted supernatants were reconstituted with specific antigen. PMN-MIF isolated by polyacrylamide gel electrophoresis showed the same properties as PMN-MIF present in whole supernatants. This factor consistently migrated in the albumin region on gel electrophoresis. These data indicate that human PMN-MIF is antigen-dependent. INTRODUCTION Sensitized lymphocytes exposed to specific antigen elaborate a variety of humoral mediators, including migration inhibitory factor (MIF). Both macrophages and polymorphonuclear leucocytes have been used as indicator cells in assay systems designed to detect MIF. However, recent studies (Rocklin, 1974) have suggested that the mediator that inhibits macrophage motility is different from that affecting migration of polymorphonuclear leucocytes. In the study reported here, these factors are referred to as macrophage MIF (M-MIF) and polymorphonuclear leucocyte MIF (PMN-MIF). Although specific antigen is known to induce production of these inhibitory factors, there is controversy as to whether their activities require the presence of antigen. Studies by David (1969) using BCG and by Yoshida, Janeway and Paul (1972) using dinitrophenyl-protein conjugates have suggested that the activity of guinea-pig M-MIF is antigen-independent. Other investigators using PPD have demonstrated antigen-dependent properties for guinea-pig M-MIF (Amos and Lachmann, 1970; Bennett and Bloom, 1967; Svejcar, Johanovsky and Pekarek, 1966). PMN-MIF has been studied less extenCorrespondence: Dr Leonard S. Goldberg, Department of Medicine, UCLA School of Medicine, Los Angeles,

California 90024, U.S.A.

223

R. H. Welsbart et al. 224 sively. Work by Clausen (1973) suggested that the activity of human PMN-MIF does not require the presence of antigen. By contrast, a recent report from our laboratory has indicated that the activity of human PMN-MIF is antigen-dependent (Weisbart, Cunningham, Bluestone and Goldberg, 1973). Using a modified agarose technique, we showed that supernates containing PMN-MIF lost all activity when diluted 1:50 with medium; when the diluted supernatants were reconstituted with antigen, MIF activity was restored. In this report we extend our initial observations to show that PMN-MIF, isolated by polyacrylamide gel electrophoresis, is antigen-dependent.

MATERIALS AND METHODS Antigens PPD containing 50,000 tuberculin units per milligram was generously provided by Dr H. B. Delvin (Parke-Davis Company, Detroit, Michigan). Coccidioidin (lot number XV B67F), was a gift from Dr Milton Huppert, Veterans Hospital, Long Beach, California; the antigen was prepared from twenty-four strains of Coccidioides immitis. Candida antigen (Hollister Stear) was prepared by water extraction and contained 20,000 PNU per millilitre. This preparation was extensively dialysed against Hanks's buffered salt solution (BSS), diluted 1:20 with Hanks's BSS, and passed through a 0-45 gtm Millipore filter.

Lymphocyte separation and culture techniques Lymphocytes were isolated from peripheral blood as previously described (Weisbart, Webb, Bluestone and Goldberg, 1972). After the cells were washed with Hanks's BSS, lymphocytes were cultured in the presence and absence of PPD, 250 tuberculin units per millilitre, or coccidioidin, 0-1 ml of a 1: 1000 dilution per millilitre, as follows: 106 lymphocytes were cultured for 7 days at 370 in 12 x 75 mm polypropylene tubes containing 1 ml of medium 199 with 25 mm Hepes buffer and 2-2 g of NaHCO3/litre (Grand Island Biological Company, Berkeley, California), 10 per cent horse serum, penicillin (100 u/ml) and streptomycin (100 pg/ml). After 7 days the tubes were centrifuged for 10 minutes at 2000 g and the supernatants were decanted. Cells were viable at 7 days as determined by trypan blue exclusion. Following termination of the cultures, non-stimulated, i.e. antigenfree, supernatants were reconstituted with the same concentration of antigen as was used in the stimulated cultures.

Agarose gel technique Preparation of agarose medium. PMN-MIF was detected by an agarose gel technique (Weisbart et al., 1973). A 2 per cent solution of agarose in medium 199 was prepared daily. Horse serum inactivated at 560 for 30 minutes was prewarmed to 470 and mixed with medium 199 prewarmed to 470 to provide a final concentration of 10 per cent horse serum and 1 per cent agarose. Penicillin (100 u/ml) and streptomycin (100 Yg/ml) were added and the pH was adjusted to 7-3. The medium was passed through a 0-45 Ym Millipore filter with a prefilter. Five-millilitre aliquots of agarose were pipetted into 60 x 15 mm plastic culture dishes and allowed to gel. Eight wells (3 mm) were cut in each plate. Preparation of leucocyte indicator cells. Leucocytes from subjects who gave negative skin test reactions to PPD, coccidiodin, or Candida were used as indicator cells. Venous blood was collected in siliconized glass tubes containing preservative-free heparin (1 per cent);

Polymorphonuclear Leucocyte Migration Inhibitory Factor 225 the red blood cells were sedimented with 6 per cent dextran 250 in the ratio 25 parts of blood to 9 parts of dextran for 45 minutes. The leucocytes were washed three times with Hanks's BSS and were resuspended in medium 199. Agarose technique. Supernates from lymphocyte cultures were replenished with an equal volume of medium 199 with 10 per cent horse serum prior to testing. 7 x 106 buffy coat cells were suspended in 0-2 ml of each replenished supernate. The cell suspension was incubated at 370 for 30 minutes, and adjusted to 3 x 108 leucocytes per millilitre. Fivemicrolitre aliquots of the suspension were dispensed into four alternate wells. Tests were performed in quadruplicate on a single plate. Plates were incubated at 370 for 18 hours in a CO2 incubator. The amount of migration, excluding the area of the well, was measured with an ocular micrometer. The migration index (MI) was calculated by dividing the mean area of migration of cells exposed to antigen stimulated supernate by the mean area of migration of cells exposed to non-stimulated supernate. Standard error of the mean (s.e.m.) of the MI was computed from the equation for s.e.m. for x/y (Dahlberg, 1940). P values for the difference between the migrations produced by stimulated and nonstimulated supernates were determined by the Student's t-test. Efect of antigen on leucocyte migration. Aliquots of buffy coat cells were suspended in medium 199 containing PPD (125, 250, 500, 1000 and 2000 tuberculin u/ml). The migration of these buffy coat cells in agarose gels were simultaneously compared with the migration of buffy coat cells incubated with medium 199 alone. The same procedure was carried out with coccidioidin at the following dilutions: 1: 10; 1: 100; 1 :1000.

Experiments to demonstrate antigen dependency and antigen specificity of PMN-MIF Studies on whole lymphocyte supernatants. Lymphocytes were isolated from the blood of four subjects sensitive to PPD; two of these subjects were sensitive to both PPD and coccidioidin; one subject was sensitive to both PPD and Candida. In addition, four healthy individuals unresponsive to PPD and four subjects unresponsive to coccidioidin by skin tests were included as controls. The lymphocytes were cultured for 7 days in the absence (non-stimulated) and presence (stimulated) of PPD or coccidioidin. The non-stimulated and PPD-stimulated supernatants were tested for PMN-MIF activity and subsequently diluted 1:50 with medium 199; in previous studies with PPD, PMN-MIF activity was shown to disappear at a dilution of 1: 50. An equal amount of fresh medium with serum was added to 0-1 ml of each diluted supernate; these were assayed for MIF before and after reconstitution with 20 tuberculin units of PPD, or 25 pl of a 1: 3000 dilution of coccidioidin. Similarly, non-stimulated and coccidioidin-stimulated supernatants were assayed for MIF before and after dilution (1:50) with medium 199. The diluted supernates were reassayed for MIF before and after reconstitution with PPD or coccidioidin. In addition, the following experiment was performed to determine whether antigen in non-stimulated supernates affected the migration of buffy coat cells. The diluted (1: 50), non-stimulated supernates were reconstituted with PPD and coccidioidin. Aliquots of buffy coat cells were simultaneously incubated with these supernates and with medium 199 alone for 30 minutes at 370, and they were assayed for MIF as described above. Studies on partially isolated PMN-MIF. PMN-MIF was isolated using polyacrylamide gels prepared by the method of Remold, Katz, Haber and David (1970) except that riboflavin (0-4 mg) and bis-acrylamide (0-5 g/100 ml) (upper gel) were used. Glass tubes, 0 5 x 9 0 cm, served as gel-containing columns. The gel consisted of three parts: the running gel consisted of 1 3 ml of gel solution; the spacer gel consisted of 0-3 ml; the sample

R. H. Weisbart et al. 226 gel consisted of 0-1 ml. After gel polymerization, 50 y1 of non-stimulated and 50 Jd of PPD-stimulated supernates obtained from five subjects were layered onto separate gel columns and subjected to electrophoresis. A current of 2-0 mA per column was applied for 90 minutes. A separate gel containing medium 199 and 10 per cent of horse serum was electrophoresed and stained with amido black. The gels were sectioned into three equal portions: section A contained albumin; section B contained the beta-globulins; section C contained the gamma-globulins. Gels were eluted at 40 by electrophoresis (Remold et al., 1970), and the eluates were dialysed for 24 hours at 40 against three changes of Hanks's BSS. The volume of eluate was adjusted to 0 3 ml with Hanks's BSS and stored at -200 until assayed for PMN-MIF. In separate experiments, the eluates were diluted 1: 10 with medium and tested for PMN-MIF before and after reconstitution with PPD, coccidioidin, or Candida as described above. The concentration of Candida antigen used for reconstitution (25 p1 of a 1:60 dilution) was that previously shown to stimulate production of PMN-MIF by sensitized lymphocytes. Dialysability. Five eluates were prepared from polyacrylamide gels and shown to contain PMN-MIF activity. The eluates were dialysed (2-4 nm pore size) against three changes of 100 x the volume of Hanks's BSS for 24 hours and reassayed for PMN-MIF.

RESULTS EFFECT OF ANTIGEN ON LEUCOCYTE MIGRATION

The migrations of buffy coat cells incubated with medium alone and with various concentrations of PPD and coccidioidin were assayed by the agarose method. The MI for each concentration is shown in Table 1. No significant inhibition of migration was obTABLE 1 EFFECT

Antigen PPD

Coccidioidin

OF ANTIGEN ON LEUCOCYTE MIGRATION

Concentration

Migration index + s.e.m.

125* 250 500 1000 2000

1.00+010 1*06+0*10 100+0-08 0-89+0-80

1: 1000t

1.00 + 0 00

1:100 1:10

0-77+0-081 1-00+0-00 1[00+0-00

* Tuberculin units per millilitre.

t Dilution in Hanks's solution.

$ P< 0 05 differs from control migration.

served at concentrations of PPD up to 1000 tuberculin units/ml. Only at a concentration of 2000 tuberculin units/ml was inhibition of migration evident (MI = 0-77+0-08, P< 0 05). This concentration of PPD (2000 tuberculin units/ml) was sixteen times greater than the amount of PPD present in the replenished lymphocyte supernates used in the dilution-reconstitution experiments. Similarly, no inhibition of migration was noted with coccidioidin at concentrations as great as 1:10, or one hundred times that present in replenished lymphocyte supernates.

Polymorphonuclear Leucocyte Migration Inhibitory Factor

227

EXPERIMENTS ON ANTIGEN DEPENDENCY OF PMN-MIF

Studies on whole supernates The results of the experiments performed on patients D.F. and W.P. who were sensitive to PPD and coccidioidin are listed in Tables 2 and 3. Supernates collected from lymphocytes exposed to PPD contained MIF activity with MI of 0-79+0 03 (s.e.m.) (D.F.) and 0 83+0 00 (W.P.) (Table 2). When the supernates were diluted 1:50, MIF activity was TABLE 2 ANTIGEN DEPENDENCY OF PMN-MIF IN WHOLE PPD-STIMULATED

SUPERNATANTS

Subjects* Test sample

Supernatant§ Supernatant diluted 1:50 Supernatant diluted 1: 50+ PPD *

D.F.

W.P.

0 79 + 0 03t 0-95 + 0 03 0-87 + 0.04t

0-83 + 0°00t 1L00 + 007 0-61 + 0-04t

Results expressed as migration index+ s.e.m.

t P< 0-01 differs from control migration. t P< 0 05 differs from control migration.

§ Supernatant collected from lymphocytes cultured with PPD for 7 days.

lost. Following reconstitution with PPD, activity was restored in the supernatant from D.F. (MI = 0 87+0 04) and W.P. (MI = 0-61+0-04). Reconstitution of the PPDstimulated supernatants with coccidioidin failed to restore MIF activity (MI = 1-03 + 0 04 and 1 02+0.04). Table 3 shows the results of reconstitution experiments with the cocciTABLE 3 ANTIGEN

DEPENDENCY OF

PMN-MIF

IN WHOLE

COCCIDIOIDIN-STIMULATED SUPERNATANTS

Subjects* Test sample

Supernatantt

Supernatant diluted 1: 50 Supernatant diluted 1:50 + coccidioidin

D.F.

W.P.

0 73 + 0-02t 100+ 0-08 0-77 + 0 02t

0-76 ± 0-05t 1 03 + 005 0-79 + 0-03t

* Results expressed as migration index+ s.e.m. t P< 0 01 differs from control migration. $ Supernatant collected from lymphocytes cultured with coccidioidin for 7 days.

dioidin-stimulated supernatants. Both supernatants contained PMN-MIF with MI of 0 73 + 0 02 and 0 76 + 0.05. MIF activity was lost when the supernates were diluted 1: 50. Reconstitution of the diluted supernates with coccidioidin restored MIF activity in supernatant D.F. (MI = 0 77+0 02) and W.P. (MI = 0-79+0 03). The diluted coccidioidin-stimulated supernates failed to show MIF activity when reconstituted with PPD (MI = 103+0 04). In addition, the non-stimulated supernates from D.F. and W.P. were reconstituted with PPD and coccidioidin. The reconstituted supernatants were incubated with buffy coat cells and assayed by the agarose method. Buffy coat cells incubated with medium alone served as the control. The MI for supernates D.F. and W.P. reconstituted with PPD were

228 R. H. Weisbart et al. 1 00+ 00 and 1b08+0-12. The MI for supernates D.F. and W.P. reconstituted with coccidioidin were 0-98+0 09 and 1b06+ 010. Four PPD skin test-negative subjects showed no response to PPD in the agarose assay; the migration indices were 1 00+0-04 (D.P.) 1 +-00+0-04 (I.N.), 1 00+0 00 (S.M.) and 1 00+0 00 (R.W.). Similarly, the lymphocytes of our coccidioidin skin test-negative subjects gave no response to coccidioidin in vitro; the migration indices were 1-00+0-04 (E.S.), 1 02+0 04 (G.S.), 1b02+0 04 (M.S.) and 1 02+0 05 (B.P.). Studies on isolated MIF Polyacrylamide gel studies demonstrated PMN-MIF activity in the albumin fraction with no inhibitory activity in the other sections of the gel. The MI of eluates obtained from four PPD-stimulated supernates were 0-58+0 03 (D.F.), 0 74+0 04 (W.P.), 0-85+0-03 (L.S.) and 0-78+0-03 (B.S.) (Table 4). Both PPD and coccidioidin antigens were shown to be heterogeneous when electrophoresed on polyacrylamide gels; however, both antigens were present in the albumin region. This explains why eluates of the albumin portion of the gel contained MIF activity without the further addition of specific antigen. MIF activity was lost, however, when the eluates were diluted 1: 10 with medium. Reconstitution with 100 tuberculin units/ml restored MIF activity in each case (MI = 0 77+0-06) 0-73+0 04, 0-86+0 04 and 0-85+0 04). Reconstitution of these eluates with coccidioidin (D.F. and W.P.) or Candida (B.S.) failed to restore MIF activity (MI = 0-96+ 0 07, 1 00+0-08 and 0 97+0 03). Similar results were obtained from gel fractions containing PMN-MIF stimulated by coccidioidin (Table 5). The MI were 0-81 +0 04 (D.F.), 0-77+0-06 (W.P.) and 0-72+0-03 (A.L.). MIF activity was lost when the eluates were diluted 1: 10 with medium. Reconstitution with coccidioidin restored the MIF activities (MI = 0-77+0-06, 0-82+0-05 and 0-84+0-03). Reconstitution with PPD (D.F. and W.P.) and Candida (A.L.) failed to restore MIF activity (MI = 1-00+0-04, 0-98+0 02 and 1.00+0.04). Heat stability MIF tests were performed on four PPD-stimulated supernatants before and after heating at 560 for 30 minutes. MI before heating were 0-76+0.00 (L.S.), 0-84+0.06 (D.F.), 0-74+0-04 (B.S.) and 0-61+0-00 (N.H.). After heating, MI were 0-80+0-03 (L.S.), 0-88+0-04 (D.F.), 0-82+0 04 (B.S.) and 0 79+0 03 (N.H.). All MI were significant at P< 0 05. No alteration in MIF activity resulted from heating at 560 for 30 minutes. TABLE 4 ANTIGEN DEPENDENCY OF PMN-MIF ISOLATED FROM PPD-STIMULATED

SUPERNATANTS

Subjects* Test sample

Eluate§ Eluate diluted 1:10 Eluate diluted 1: 10 + PPD

D.F.

W.P.

L.S.

B.S.

0-58 _ 0-03t 100+0 04 0 77 + 0.(6$

074 + 0-04t

0-85 + 0-03t 0-98+0 04 0-86 + 0 04t

0-78 + O-03t 100O+004 0 85+0 04$

0-96+0-06

0 73 + 0-04t

* Results expressed as migration index + s.e.m. t P < 0-01 differs from control migration. P < 0 05 differs from control migration. § Supernates from lymphocytes cultured with PPD for 7 days were subjected to polyacrylamide gel electrophoresis. Eluate obtained from the albumin region of the gel contained MIF activity.

Polymorphonuclear Leucocyte Migration Inhibitory Factor

229

TABLE 5

ANTIGEN

DEPENDENCY OF

PMN-MIF

ISOLATED FROM COCCIDIOIDIN-STIMULATED SUPER-

NATANTS

Subjects* Eluate§ Eluate diluted 1:50 Eluate diluted 1:50 + coccidioidin

D.F.

W.P.

A.L.

0-81 + 0-04t

0 77 + 0-061 1b00+0 04 0-82 + 0-05t

0-72 + 0-03t 100O+004

1-00+0-08 0 77 + 0-06$

0-84 + 003t

* Results expressed as migration index + S.E.M. t P< 0 01 differs from control migration. t P < 0-05 differs from control migration. § Supernatants from lymphocytes cultured with coccidioidin for 7 days were subjected to polyacrylamide gel electrophoresis. Eluate obtained from the albumin region of the gel contained MIF activity.

Dialysability PMN-MIF was recovered from polyacrylamide gels and dialysed against Hanks's BSS. MIF produced in response to PPD was found in the dialysed samples with MI of 0 58 + 0 03 (D.F.), 0 74+0 04 (W.P.), 0-85+0 03 (L.S.) and 0-78+0-03 (B.S.). MIF produced to coccidioidin was detected in the eluates of D.F., W.P. and A.L. with an MI of 081 +0-04, 0 77+0-06 and 0-72+0-03. These results were significant at P

Human polymorphonuclear leucocyte migration inhibitory factor. Evidence for antigen dependency.

Immunology, 1975, 29, 223. Human Polymorphonuclear Leucocyte Migration Inhibitory Factor EVIDENCE FOR ANTIGEN DEPENDENCY R. H. WEISBART, JUDITH ISAAC...
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