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E. Pick, Y. Godny and E.F. Gold

Eur. J. Immunol. 1975.5: 584-587

5. References

14 Campbell, A.C., Skinner, J.M., Hersey, P., Roberts Thomson, R., McLennan, I.C.M. and Truelove, S.C., Clin. Exp. Immunol. 1974. 16: 521.

1 McLennan, I.C.M., Transplant. Rev. 1972. 13: 67. 2 Biberfeld, P., Biberfeld, G., Perlmann, P. and Holm, G., Cell. Immunol. 1973. 7 : 60. 3 Wisldff, F., FrQland, S.S. and Michaelsen, T.E., Int. Arch. Allergy Appl. Immunol. 1974.47: 139. 4 FrQland, S.S., WislQff,F. and Michaelsen, T.E., Int. Arch. Allergy Appl. Immunol. 1974.47: 124. 5 Greenberg, A.H., Hudson, L., Shen, L. and Roitt, I.M., Nature-New Biol. 1973.242: 111. 6 Editorial comment, Nature-New Biol. 1973.242: 229.

15 Du Bois, M.J.G.J., Huismans, D.R., Schellekens, P.Th.A. and Eijsvoogel, V.P., Tissue Antigens 1973.3: 402. 16 Bdyum, A., Scand. J. Clin. Invest. 1968.21: 1. 17 Weening, R.S., Roos, D. and Loos, J.A., J. Lab. Clin.Med. 1974. 83: 570.

Du Bois, M.J.G.J., Schellekens, P.Th.A., Huismans, D.R. and Raat-Koning, L., Publication des colloques de l'tNSEW, Lyon 1973, p. 101.

18 Eijsvoogel, V.P.,

7 Wisldff, F. and Frdland, S.S., Scand. J. Immunol. 1973.2: 151.

19 Cerottini, J.C., Engers, H.D., McDonald, H.R. and Brunner, K.T., J. Exp. Med. 1974.140: 703.

8 Greenberg, A.H., Shen, L. and Roitt, I.M., Clin. Exp. Immunol. 1973.15: 251.

20 Zighelboim, J., Gall, R.P., Ossorio, R.C. and Fahey, J.L., Transplantation 1974. 18: 27.

9 Trinchieri, G., Bernoco. D., Curtoni, S.E., Miggiano, V.C. and Ceppellini, R. in Dausset, J. and Colombani, J. (Eds.), Hisrocompatibility Testing, Munksgaard Copenhagen 1973, p. 509.

21 Zighelboim, J., Bonavida, B. and Fahey, J.L., J. Immunol. 1973. 111: 1737.

10 Campbell, A.C., Hersey, P., McLennan, I.C.M., Kay, H.E.M. and Pike, M.C.,Brit. Med. J. 1973.2: 385. 11 Holm, G. and Hammarstrom, S., Clin. Exp. Immunol. 1973. 13: 29. 12 Gelfand, E.W., Resch, K. and Presser, M., Eur. J. Immunol. 1972. 2: 419. 13 Campbell, A.C., McLennan, I.C.M., Snaith, M.L. and Barnett, I.G., Clin. Exp. Immunol. 1972. 12: 1.

22 Holm, G., Int. Arch. Allergy Appl. Immunol. 1972.43: 671. 23 Larsson, A., Perlmann, P. and Natvig, J.B., Immunology 1973. 25: 675. 24 McLennan, I.C.M., Howard, A., Cotch, F.M. and Quil, P.G., Immunology 1973.25: 459. 25 Wisldff, F., Michaelsen, T.E. and FrQland, S . S . , Scand. J. Immunol. 1974.3: 29. 26 Abramson, N. and Schur, P.H., Blood 1972.40: 500.

Short Paper E. Pick, Y. Godny and E.F. Gold Department of Human Microbiology, Tel-Aviv University Medical School, Tel-Aviv

Participation of immunoglobulin-bearing lymphocytes in the production of macrophage migration inhibitory factor T h e role o f immunoglobulin-bearing cells in t h e production of macrophage migration inhibitory factor ( M I F ) b y tuberculin-stimulated lymphocytes of guinea pigs, immunized with complete Freund's adjuvant, was studied. It was found that: ( I ) pretreatment o f lymphocytes with rabbit anti-guinea pig IgG (anti-IgG) does not block antigen-induced MIF production. ( 2 ) I'as sage o f lymphocytes through double layer IgG-anti-lgG gelatin bead columns ( t h e preparation of which is described) abolishes MIF formation b y the eluted cells. Cells retained o n t h e columns can b e recovered and where shown t o produce MIF, when stimulated b y antigen. (3) Pulsing of lymphocytes with antiIgC, f o r 2 h a t 37 O C , results in MlF synthesis b y t h e cells cultured in medium, in t h e absence of specific antigen. These findings indicate that cells bearing Ig or Ig fragments are either able t o secrete MIF themselves, u p o n stimulation with antigen o r anti-lgG, o r are required f o r MIF production by a different cell type.

1. Introduction

Macrophage migration inhibitory factor (MIF) is t h e best characterized lymphokine produced by activated lymphocytes in vitro. M I F synthesis can b e induced b y stimulating lymphocytes with specific antigen [ 1, 21 with nonspecific mitogens like concanavalin A [3] and phytohemagglutinin [4], with anti-lymphocyte serum [4]and with antigen-antibody complexes [S]. [I 9781 ~

~~~

Correspondence: Edgar Pick, Department of Human Microbiology, Tel-Aviv University Medical School, Ramat-Aviv, Tel-Aviv, Israel Abbreviations: MIF: Migration inhibitory factor CFA: Complete Freund's adjuvant PPD: Tuberculin purified protein derivative LNC: Lymph node cells MEM: Eagle's minimum essential medium PEC: Peritoneal exudate cells

Little is known a b o u t t h e nature o f MIF-producing lymphocytes b u t it was generally assumed that they belong t o the thymus-dependent ( T ) population. T h e arguments for this were: t h e good correlation between cell-mediated immunity and MIF production, t h e finding that spleen cells of thymectomized a n d irradiated chicken d o not release MIF while cells from bursectomized birds d o [ 6 ] , t h e report that monocyte chemotactic factor production is normal in agammaglobulinemic chicken [7] and t h e finding that lymphocytes responsible for t h e h"F-mediated macrophage disappearance reaction lack complement receptors a n d therefore belong t o t h e T class [8]. O n t h e o t h e r hand, several recent reports indicate that, under certain conditions of stimulation, B cells are equally able t o secrete lymphokines [9-1 I]. It has also been found that Ig-producing human lymphocyte lines release MIF [ 121. Tuberculin purified protein derivative (PPD) is the most widely used antigen for t h e specific induction of MIF formation. In

Eur. J. Immunol. 1975.5: 584-587

Immunoglobulin-bearing lymphocytes and MIF production

order t o gain information about t h e nature of t h e cells responsible for antigen-induced MIF synthesis, we have examined: (a) the influence of preincubating guinea pig lymphocytes with anti-IgG antibody o n MIF production in response t o PPD stimulation, and (b) t h e effect of passing lymphocytes of PPD-sensitive animals through columns of gelatin beads coated with anti-IgG, on their capacity t o synthesize MIF upon PPD stimulation. We found that: (1.) MIF production by specifically sensitized lymphocytes exposed t o PPD is not blocked by pretreatment of the cells with anti-IgG. (2.) AntiIgG immunoabsorbent columns eliminate cells which are essential for PPD-induced MIF secretion and these cells can b e subsequently recovered. In t h e course of these experiments it was also found that MIF synthesis is regularly induced by pulsing lymphocytes with anti-IgC.

of an ice-cold solution of guinea pig IgG ( 2 mg/ml) in phosphate buffered saline, pH 7.2 (PBS), which was continuously stirred SO as to assure the dispersion of the gelatin as discrete particles. Of a 2.5 % solution of glutaraldehyde in PBS, 40 ml was then added dropwise t o the gelatin suspension, stirred magnetically a t 4 ‘C. After all t h e glutaraldehyde has been added, the mixture was stirred for 1 h a t 4 ‘Cand allowed to stand undisturbed overnight in the cold. The unattached IgG was then removed by transferring the conjugated gelatin t o a large funnel covered with a nylon cloth screen with 37 p sized pores (“Nytal”, type 445, Swiss Bolting Cloth Mfg. Co., Thal, Switzerland) and washing it with 4 liters of ice-cold PBS. T h e beads were suspended in cold MEM and transferred t o glass columns (2.5 x 1 1 cm) plugged a t the lower end with glass wool. Each column contained the equivalent of 5 g dry gelatin powder. The columns were subsequently washed with 50 ml PBS and filled with 30 m l of undiluted whole rabbit anti-guinea pig IgG, which displaced all the PBS. Control columns were filled with t h e same volume o f normal rabbit serum (NRS). They were allowed t o stand f o r 2 h at 4 OC and then washed in the cold with a succession of 1SO ml PBS and 150 ml MEM. LNC were brought t o a concentration of 10’viable cells/ml and a total of 3 x 10’ - 5 x 10’ cells applied per column and allowed t o enter the gelatin bead bed. Elution was performed at 4 OC with 40 ml of MEM at a flow of 2 ml/min. When a succession of two columns was used t h e cells eluted during the first passage were centrifuged, brought t o a volume of 3-5 ml and applied o n an identical second column and eluted as before. The retained cells were recovered by filling the columns t o the t o p with 20 ml MEM, containing 10 % normal guinea pig serum, and stirring u p the gelatin beads with t h e aid of a pipette.

2. Materials and methods 2.1. Procedure

Male Hartley guinea pigs were injected in t h e footpads with 1 ml complete Freund’s adjuvant (CFA, Difco, Detroit, Mich., USA), containing 0.5 mg M. tuberculosis H37Ra. Lymph node cells (LNC) were harvested 4 weeks later and purified by glass bead filtration a t 37 OC, in t h e presence of 20 % calf serum, as previously described [ 131. The cells were cultured in serumfree Eagle’s minimum essential medium (MEM) for 20 h at 37 OC, in plastic tissue culture flasks (Falcon Plastics, Los Angeles, Calif.), in a n atmosphere of 90 % air and 10 % COz. MlF in supernatants was assessed by the capillary migration inhibition test, using oil-induced peritoneal exudate cells (PEC) of normal guinea pigs [ 131.

2.2. Preparation of guinea pig IgG and rabbit anti-IgG Guinea pig IgG was prepared from pooled serum of animals immunized with CFA, by two cycles of batch DEAE-cellulose chromatography, using a 0.01 M potassium phosphate buffer, pH 8.0 for eluting t h e IgG [ 141. The purified material gave a single precipitation line in the typical region, o n immunoelectrophoresis with rabbit anti-whole guinea pig serum. Rabbits were immunized with guinea pig IgG by three weekly injections of 10 mg protein, incorporated in incomplete Freund’s adjuvant (IFA, Difco), given in t h e footpads and multiple subcutaneous sites, followed by two boosters of 10 mg protein administered subcutaneously, without IFA. The rabbits were bled 10 days after the last injection. The IgG fraction of the rabbit antiserum was purified by batch DEAE-cellulose chromatography [ 141. Rabbit anti-guinea pig IgG (anti-lgG) gave a single precipitation line with pooled guinea pig serum subjected t o immunoelectrophoresis. The antiserum was also tested for an eventual antilymphocyte activity. Serial dilutions of anti-IgG were incubated with guinea pig thymocytes for 1 h at 37 ‘C in the presence and absence of complement. No agglutination o r complement-dependent cytotoxicity (as assessed by trypan blue exclusion) was detected. 2.3. Anti-IgG immunoabsorbent columns

Anti-IgG immunoabsorbent columns were prepared by t h e double layer principle. First, gelatin beads were coated with guinea pig IgG, as described b y Gold, Kleinman and BenEfraim [ 151 and subsequently overlayered with rabbit antiguinea pig IgG in excess, as described by Wigzell, Sundquist and Yoshida [ 161. The detailed procedure was as follows: 20 g gelatin powder (Bacto Gelatin, Difco) were suspended in 400 ml

585

The stopcock was then opened and the detached cells allowed to drain at maximum speed. This procedure was repeated once more. All eluted cells were centrifuged, washed once with M E M and brought t o a concentration of IO’viable cells/ml. We would like t o emphasize the fact that both specific and control columns contained IgG-coated beads and only the second layer was different (anti-lgG o r NKS). The efficiency of double layer gelatin anti-lgG immunoabsorbents, prepared as describcd above, was confirmed by the nearly complete retention by such columns of sheep red cells coated with guinea pig IgG.

3. Results and discussion

Exposure of LNC from PPD-sensitive guinea pigs t o a 2-h pulse with rabbit anti-guinea pig IgG, followed by the removal of anti-IgG by washing, did not impair their ability t o produce MIF upon incubation for 20 h with PPD (Table I ) . On the contrary, anti-IgG-pulsed lymphocytes produced slightly more MIF, after PPD stimulation, than control cells, pulsed with NRS. It has been recently reported that treatment of human peripheral blood lymphocytes with anti-F(ab’)z [ 171 o r anti-light chain antibody [ 181 abolished the PPD-induced inhibition of migration in a direct migration assay, involving a mixture of human lymphocytes and guinea pig PEC. In both reports no blocking of MIF production was seen after pretreatment with anti-?, anti-p and a n t i 4 chain sera [17, 181. While this article was being prepared it was reported that antigen-induced M I F production by guinea pig LNC was little influenced by pretreatment of the cells with anti-lgGZ or anti-lgG1 antiserum, but partially suppressed by anti-light chain antiserum [ 191. The main disadvantage of the pulse technique is that a hypothetical membrane-bound anti-lgG-IgG complex could be in-

E. Pick, Y. Godny and E.F. Cold

586

Eur. J. Immunol. 1975.5: 584-587

Table 1. Effect of pulsing lymphocytes with anti-lgG on PPD-induced MIF production inhibition of migration by supernatant of LNC stimulated with PPLY) pulsed with pulsed with NR S ~ ) anti-IgO)

%'

Iixp. No.

Material for pulsing and concentration Whole serum 1 / I 0 Whole serum 1/10 Whole serum 1/10

1

2 3 4 5 6

35.1 47.8 44.6 46.3 55.9 80.4

39.6 56.9 52.2 49.1 74.7 80.9

51.7 f 6.3

58.9 2 6.4

Whole serum 1/10 Purified IgC 1 mg/ml Purified IgC 0.5 mg/ml Mean f S.E.

As it appears from Table 2, passage of LNC through anti-lgG columns reduced considerably o r suppressed totally the PPDinduced MIF production. Passage of cells through columns of IgG-coated beads reacted with NRS leaves MIF production intact. LNC retained by t h e anti-IgG columns could be recovered in considerable number and in viable form, as described under Section 2.3. These lymphocytes had an MIF producing potential identical t o that of t h e initial cell population applied t o t h e column. These findings can be interpreted as indicating either that: (a) lymphocytes producing MIF in response t o PPD, possess surface Ig o r at least immunoglobulin light chains, or (b) an Ig-bearing cell is required for M I F production b y a different cell, not possessing surface Ig. This second possibility would imply some form of cell cooperation in MIF formation, as proposed in the past b y Pick and Turk [ 51.

The possibility that Ig-bearing cells might actually secrete MIF was tested b y exposing guinea pig LNC to a 2-h pulse of anti% inhibition of IgG serum o r its purified IgG fraction. Control cells were = 100 migration pulsed with identical concentrations of NRS or NRS IgG. As seen in Table 3, pulsing with anti-IgC, followed by culture in MEM, resulted in t h e release o f MIF-like activity into t h e superSupernatants from each experiment were tested on two separate 0 ~ - natant. More potent supernatants were obtained when the concasions on distinct batches of target PEC. The values represent means centration of cells in culture was raised from lo7 cells/ml t o derived from the results of these two tests. LNC (107 viable cells/al) were incubated for 2 h, at 37 O c with either 2 X l o 7 CellS/ml. Production Of the inhibitory material Was whole anti-IgC or the IgG fraction of anti-lgG diluted in Eagle's MEM. abolished by culturing the anti-IgG-pulsed cells in the presence Control cells were incubated with an identical dilution of NRS or the of 10 pg/ml puromycin, suggesting an active, protein synthesis same concentration of NRS IgG. After completion of the pulse, the requiring process. This phenomenon is similar to the induction cells were spun down, washed two times with medium, adjusted to a concentration of 107 viable cells/ml in Eagle's MEM and cultured for of MIF Secretion b y short-term exposure of guinea pig lymphoThe identi20 h with and without PPD (50 pglml). Supernatants of lymphocytes cytes t o heterologous anti-lymphocyte serum [4]. incubated in the absence of PPD, were supplemented with 50 p g h l fication of the anti-lgG-induced inhibitory material as M I F PPD, after culture. must await its chemical characterization. mean area of macrophage migration in supernatant of PPDstimulated lymphocytes (after NRS or anti-IgG pulse) mean area of macrophage migration in supematant ofunStimulated lymphocytes (after NRS or anti-IgG pulse)

gested by the cell o r shed into the medium, processes which were found t o be followed by the reappearance of membrane immunoglobulin in increased concentration [ 201. In order t o circumvent this possibility we made use of specific immunoabsorbent columns able t o retain lymphocytes possessing IgG o n their surface. In order t o achieve a high density of anti-IgG o n the insoluble matrix, we first coated the gelatin beads with pure IgG, this being followed b y the specific binding of anti-IgG in excess [ 161. This type of column was found to retain only cells carrying t h e specific immunoglobulin towards which the second layer antibody was directed. Cells merely possessing receptors for Fc were not retarded o n such columns, unless they were also immunoglobulin positive [ 161.

4. Concluding remarks

The findings reported in this article demonstrate that, in the guinea pig, Ig-bearing lymphocytes are able t o produce MIF, when stimulated with anti-IgC, and are required for MIF production induced b y PPD. From the available data it is not possible t o determine whether cells producing MIF upon pulsing with anti-IgG are identical with those producing MIF after PPD stimulation. The possibility must also be considered that the double layer IgG-anti-IgG columns will retain not only Ig-bearing cells but also lymphocytes with F c receptors. In the mouse this latter

Table 2. Depletion and recovery of MIF-producing cells after filtration of LNC through IgC-anti-IgG immunoabsorbent columns

Exp. no.8) 1 2 3

Unfractionatcd Cells passed through cells I&-NRS columns % inhibition % inhibition 'J viable cells of migration rccoveredc) of migrationb) 40.4 46.0 50.8

61.3 45.2 48.0

51.9 54.5 41.3

Cells passed through I&-anti-IgG columns % inhibition 70 viable cells of migration recovered 37.3 22.4 16.0

31.4 24.7 - 9.4

Cells bound to and recovered from IgC-anti-IgC columns %viable cells % inhibition recovered of migration - d) 38.0 40.2

- d)

52.1 52.1

In Exp. no. 1, the cells were passed through one IgG-anti-lgG column only while in Expts. No. 2 and 3, a succession of two such columns was used. In all experiments control cells were passed through an identical number of IgG-NRS columns.

mean area of macrophage migration in supernatant of PPD-stimulated lymphocytes x 100 mean area of macrophage migration in supernatant of unstimulated lymphocytes Supernatants from lymphocytes obtained in each cell fractionation experiment were tested on three separate occasions, on distinct batches of target PEC. The values represent means derived from the results of these individual tests. This is expressed as the percentage of viable cells recovered from the total of viable cells initially applied on the first immunoabsorbent column. In Exp. no. 1 no attempt was made to recover the cells retained on the IgG-anti-IgG column. % inhibition of migration = 100 -

Eur. J. lmmunol. 1975.5: 584-587

Immunoglobulin-bearinglymphocytes and MIF production

Table 3. Inhibition of macrophage migration by culture supernatants of lymphocytes pulsed with anti-IgG

the fact that in our experiments lymphocytes were obtained at a later time after immunization. Care must also be exercised when extending these findings t o other animal species; in t h e rabbit an anti-Fab antiserum, causing DNA synthesis in LNC, did not induce M I F production [26].

5% inhibition

Exp. no. 1 2 3 4 5

Pillsed witha) Anti-lgG Anti-lgC 1gG fraction of anti-lgC 1gG fraction of anti-lgG IgG fraction of anti-1gC Mean f S.E.

of migration by supernatants of antiConc. of Cell wnc. IgG-pulsed LNC') antiserumb) in culture 1/10 1/10 1 1

0.5

107 1.0~107 1.5~107 2 xi07 2 x10' LOX

23.5 34.2 38.1 47.0 41.5

36.9 f 3.9

a) LNC (107 viable cells per ml) were incubated for 2 h at 37

587

While this article was being reviewed it has been reported that, in humans, both T and B cells make MIF in response t o PPD but B cells are more efficient producers [27]. An important conclusion, t o be drawn from these results, is that t h e indiscriminate use of macrophage migration inhibition as an indicator of T cell-mediated immunity is not warranted. This work was supported b y the Advancement of Mankind Foundation, b y the World Health Organization and by NIH research grant No. IROI AI I I 194-01AI A L Y from the National Institute o f Allergy and Infectious Diseases. We thank Mr. D.B. Lee, from the Ministry of Ap'culture's Central Veterinary Laboratory, Weybridge, England, for generous gifts of PPD and Ms. Julia Schwartz for help in the preparation of the manuscript.

O C with either whole anti-IgC or the IgG fraction of anti-lgC, diluted in Eagle's MEM. Control cells were incubated with an identical dilution of NRS or the same concentration of NRS 1gC. The pulsed cells were centrifuged, washed two times with medium and adjusted to the cell concentration indicated in the table. Received December 11, 1974, in revised form April 1, 1975. b) This indicates the final dilution (for whole antiserum) or final protein concentration (mglml) (for purified IgG) used for pulsing. mean area of macroDhage migration in 5. References supernatant of anti-igGTpulsed LNC c) % inhibition of = x 100 migration mean area of macrophage migration 1 Bloom, B.R. and Bennett, B., Science 1966. 80: 153. in supernatant of NRS-created LNC 2 David, J.R., Proc. Nat. Acad. Sci. US 1966. 56: 72. Supernatants from each pulse experiment were tested on two or 3 Pick, E., Brostoff, J., Krejci, J. and Turk, J.L., Cell. Immunol. three separate occasions on distinct batches of target PEC. The 1970. 1 : 92. values represent means derived from the results of these individual tests. 4 Lamelin, J.P. and Vassalli, P., Nature 1971. 229: 426. 5 Pick, E. andTurk, J.L., Immunology 1972. 22: 39. category includes b o t h B and T lymphocytes [21, 221. While Morita, C. and Soekawa, M., Poultry S d 1972. 51: 1133. 6 it can be assumed that t h e presence of Fc receptors on Ig7 Altman, L.C. and Kirchner, H., J. Immunol. 1972. 109: 1149. bearing cells would increase t h e strength of their attachment to the IgC-anti-IgG columns, there are several arguments against 8 Sonozaki, H. and Cohen, S . , Cell. Immunol. 1972.3: 644. the assumption that such columns will also retain Ig-negative 9 Yoshida, T., Sonozaki, H. and Cohen, S . , J. Exp. Med. 1973. 138: cells possessing F c receptors, for instance, activated T cells. 784. These arguments are: ( I ) Wigzell et al. [ 161 have shown that 10 MacDermott, R.P., Rocklin, R.E., Chess, L., David, J.R. and IgG-anti-IgC columns function exclusively through combinaSchlossman, S.F., J. Clin. Invest. 1974. 53: 49a. tion with Ig determinants on t h e cells, since control columns 11 Mackler, B.F., Altman, L.C., Rosenstreich, D.L. and Oppcnheirn, prepared by coating the beads with bovine serum albumin J.J., Nature 1974.249: 834. (BSA) followed by anti-BSA, did not retain B cells. (2) I n 12 Tsuchimoto, T., Tubergen, D.G. and Bloom, A.D., J. Immunol. experiments performed before the development of t h e gelatin 1972. I O Y : 884. bead method, we were unable t o retain MIF producers o n col13 Manheimer, S . and Pick, E., Immunology 1973.24: 1027. umns, prepared b y t h e method of Basten et al. [23], which 14 Reif, A.E., Immunochemistry 1969. 6 : 723. are supposed t o bind cells with Fc receptors. (3) As opposed 15 Gold, E.F., Kleinman, R. and Ben-Efraim, S . , J. Immunol. Method. to the situation in the mouse [21, 221 there is, t o t h e best of 1974. 6 : 31. our knowledge, n o evidence in support of t h e assumption that 16 Wigzell, H., Sundquist, K.C. and Yoshida, T.O., Scand. J. Immunol. activated guinea pig T cells possess Fc receptors. (4) Purified 1972. I : 75. guinea pig T cells did not respond b y either DNA synthesis o r 17 Rajapakse, D.A., Papamichail, M. and Holborow, E.J., Nature-New chemotactic factor production after exposure t o antigen-antiBiol. 1973.245: 155. body complexes or aggregated Ig, t w o stimulants acting at t h e 18 Marsman, A.J.W. and van der Hart, M., Eur. J. Immunol. 1974. Fc receptor [24]. 4: 235.

The present study does not answer the question whether the Ig-bearing cell required for MIF production is a classical B cell or another cell type having acquired cytophilic Ig. It must also be emphasized that this study is limited t o one antigen only - tuberculin PPD - and that this particular antigen is known t o act as a nonspecific B cell mitogen in both mice and guinea pigs [25, 91. Our findings differ from those of Yoshida et al. [9], who found that in sensitized guinea pigs both B and T cells are able t o release MZF in response to PPD, while in our hands PPD-induced MIF production was mainly if not exclusively the attribute of Ig-bearing cells. This may be due t o the use of separation methods based o n the presence of different cell receptors or t o

19 Goscicka, T., Cell. Immunol. 1974.13: 207. 20 Loor, F., Forni, L. and Pernis, B., Eur. J. Immunol. 1972. 2: 203. 21 Yoshida, T.O. and Anderson, B., Scand. J. Immunol. 1972. I : 401. 22 Anderson, C.L. and Grey, H.M., J. Exp. Med. 1974. 139: 1175. 23 Basten, A., Sprent, J. and Miller, I.F.A.P., Nature-New BioL 1972. 235: 178. 24 Wahl, S.M., Iverson, G.M. and Oppenheim, J.J., J. Exp. Med. 1974. 140: 1631. 25 Sultzer, B.M. and Nilsson, B.S., Nature-New Biol. 1972.240: 198. 26 Pelley, R.P., Schwartz, H.J. and Stavitsky, A.B., Fed. Proc. 1973. 32: 988. 27 Rocklin, R.E., MacDermott, R.P., Chess, L., Schlossman, S.F. and David, J.R., J. Exp. Med, 1974. 140: 1303.