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Lymphocytes from guinea pigs having delayed hypersensitivity to horse-radish peroxidase (HRPO) when cultured in vitro with HRPO produce a large m.w. factor (greater than 100,000 daltons) that causes peritoneal macrophages from nonimmune animals to aggregate. The macrophage aggregation factor (MAF) can be separated from macrophage migration inhibitory factor (MIF) by gel filtration of active lymphocyte supernatants on Sephadex G-150. MAF is heat stable (56 degrees C for 30 min) but inactivated by trypsin. These data suggest that aggregation of macrophages in vitro by lymphokine-rich culture supernatants is not due to MIF but is caused by a separate large m.w. factor.
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The production of macrophage migration inhibitory factor (MIF) and immune interferon (IF) by concanavalin A (Con A)-stimulated cultures of thymus, lymph node and spleen cells was investigated. It was found that all cultures produced MIF activity, whereas only spleen cells produced marked IF activity. The capacity to produce IF was found to be correlated with the macrophage content of a cell preparation as evidenced by staining for esterase-positive cells. Furthermore, column-purified spleen T cells produced MIF but no IF. Migration inhibition caused by residual mitogen could be ruled out. On the other hand, when macrophages grown from bone marrow cells were pre-exposed to supernatants of mitogen-stimulated lymphocytes, IF activity was released into freshly added medium while no significant MIF activity was found. IF was also found in supernatants of macrophage cultures after exposure to conventional inducers in vitro (polyinosinic-polycytidylic acid, Corynebacterium parvum) or in vivo (C. parvum), whereas no MIF was detected. An anti-Type I IF serum neutralized IF in supernatants from Con A-stimulated spleen cells but did not affect MIF in the same supernatants. This indicates that IF and MIF activity are associated with different molecules. It is, therefore, concluded that under the described conditions, IF and MIF are produced by different cells. T cells are the prime producers of MIF while IF is released by macrophages following induction by lymphokines.
Guinea-pig macrophage migration inhibitory factor (MIF), obtained by the stimulation of sensitized lymph node cells with tuberculin PPD, was characterized as a glycoprotein by the following criteria: (a) its activity is destroyed by 0.02 M sodium periodate; (b) when MIF-containing culture fluids are subjected to precipitation by perchloric acid (final concentration 1 M), the inhibitory activity is recovered in the supernatant; and (c) MIF binds to Sepharose-linked concanavalin A and can be eluted with methyl-alpha-D-glucopyranoside. When MIF-containing culture supernatants are fractionated by isoelectrofocussing, migration inhibitory activity is recovered in a fraction with an isoelectric point of 4.4--4.6.
The role of immunoglobulin-bearing cells in the production of macrophage migration inhibitory factor (MIF) by tuberculin-stimulated lymphocytes of guinea pigs, immunized with complete Freund's adjuvant, was studied. It was found that: (1) pretreatment of lymphocytes with rabbit anti-guinea pig IgG (anti-IgG) does not block antigen-induced MIF production. (2) Passage of lymphocytes through double layer IgG-anti-IgG gelatin bead columns (the preparation of which is described) abolishes MIF formation by the eluted cells. Cells retained on the columns can be recovered and where shown to produce MIF, when stimulated by antigen. (3) Pulsing of lymphocytes with anti-IgG, for 2 h at 37degreesC, results in MIF synthesis by the cells cultured in medium, in the absence of specific antigen. These findings indicate that cells bearing Ig or Ig fragments are either able to secrete MIF themselves, upon stimulation with antigen or anti-IgG, or are required for MIF production by a different cell type.
Spleen cells of C57B1/6J mice immunized with complete Freund's adjuvant produced macrophage migration inhibitory factor (MIF) when incubated in vitro with tuberculin purified protein derivative (PPD). For optimal MIF production spleen cells were cultured for 48 h in a serum-free medium, at a concentration of 2 x 10(7) cells/ml. MIF was assayed in a xenogenic system, using oil-induced guinea pig peritoneal exudate cells as targets. MIF synthesis could also be induced by pulsing spleen cells for 2 h with concanavalin A, phytohemagglutinin, pokeweed mitogen or lipopolysaccharide, followed by culture in plain medium. No MIF secretion was induced by incubation of spleen cells with anti-theta or rabbit anti-mouse IgG sera. Cells producing MIF in response to PPD were characterized as B cells by virtue of being insensitive to anti-theta serum and complement, by being retained on nylon wool, glass bead and anti-Ig colums and by the presence of Fc receptors. PPD-stimulated T cells did not produce MIF. PPD-induced mouse spleen cell MIF demonstrated a moderate loss of activity by heating at 56 and 80 degrees C and was completely inactivated after digestion with chymotrypsin. By fractionation on Sephadex G-200, migration inhibitory activity was recovered in a molecular range of 100,000-12,400 daltons.
The influence of the thymus on the production of the macrophage migration inhibitory factor (MIF) was studied in C57BL and CBA mice thymectomized at 4--6 weeks of age. On the 1--21st day after the operation they were immunized intraperitoneally with complete Freund's adjuvant. MIF production stimulated by tuberculin was determined on the maximum of the immune response. MIF production was abolished in mice of both lines already during the first days. To elucidate a relationship between MIF production and the presence of the thymus the former was investigated in the thymectomized "nude" mice. The mice showed no MIF production. It was found as well that thymectomy can interrupt the immune response in early stages of its development and completely eliminates MIF production the first days after immunization. Moreover, thymectomy in adult mice also changes spontaneous migration of macrophages both in immunized and non-immunized mice. These changes were more pronounced in C57BL mice.
Treatment of guinea-pig macrophage migration inhibitory factor (MIF) containing culture supernatants with the denaturing agents guanidine hydrochloride (Gu HCl) or urea, in the presence or absence of the reducing agent 2-mercaptoethanol (2-ME), or with sodium dodecyl sulphate (SDS), does not destroy biological activity. Alkylation of reduced MIF preparations results in a considerable decrease or total loss of MIF activity. Treatment of supernatants with the combinations, Gu HCl and 2-ME or urea and 2-ME results in the recovery of MIF activity in association with molecules less than 30,000 in molecular weight (mol. wt). After removal of the agents by dialysis, MIF activity is found associated with molecules larger than 30,000. The reduction in mol. wt is dependent on the presence of 2-ME. When MIF-containing supernatants are treated with urea and SDS and fractionated by preparative polyacrylamide gel electrophoresis (PAGE) in the presence of the same agents, MIF activity is recovered in the mol. wt range of 42,000--80,000. When supernatants are treated with 2-me, in addition to urea and SDS, and preparative PAGE is performed in their presence, MIF activity is found associated with material having a mol. wt of 15,000--18,000. Analytical SDS-PAGE of this fraction reveals two or three closely grouped bands corresponding to the above mol. wt range.
Peritoneal fluid from the abdominal cavities of guinea pigs having delayed hypersensitivity to horseradish peroxidase (HRPO) was obtained by a lavage technique before and after i.p. challenge with antigen. Macrophage migration inhibitory factor (MIF) and macrophage chemotactic factor activities were measured in peritoneal fluids from each animal. Chemotactic activity for macrophages was maximal 24 hr after i.p. challenge and was absent thereafter. MIF activity was maximal in peritoneal fluid 24 to 48 hr after challenge. Macrophages were present in greatest numbers in peritoneal fluid 24 hr after challenge and returned almost to control levels at 48 hr. Macrophages in 48-hr fluid were larger and exhibited more intense cytoplasmic staining for nonspecific esterases when compared to those in 0-hr fluid. The m.w. of MIF obtained from culture supernatants of HRPO-stimulated guinea pig lymphocytes and 48-hr peritoneal fluid were found to be virtually identical, 58,000 and 54,000 daltons, respectively. MIF from these in vitro and in vivo sources were similarly resistant to heating at 56 degrees C for 30 min but were both destroyed by incubation with isoluble trypsin.
Sixty three cancer cases who received minimum of the removal of the main tumor constitute the subjects of the present study. Macrophage migration inhibitory factor (MIF) of peripheral blood lymphocytes was studied using extirpated autochthonous tumor tissues as antigen and guinea-pig peritoneal exudative cells as indicator cells immediately before the operation and the fourth postoperative week. The results indicated that in those cases of relatively early stage, i.e. Stages I and II, whose tumor was believed to have been removed completely, MIF turned negative in 7/9 (77.8 per cent) after the operation, while in others with advanced cancer of Stage IV in which the tumor bearing tissue was probably not completely removed, MIF turned positive in most cases (13/17: 75.6 per cent) postoperatively, even though it was negative before the operation. In the cases of Stage III with cancer progressed to an intermediate degree, in about half of the cases (7/11: 63.6 per cent) MIF turned negative after the operation and in the other half (7/10: 70 per cent) MIF became positive postoperatively, suggesting that for MIF to persist the presence of certain amount of tumor tissue is necessary.
High immunological specificity of the direct and indirect macrophage migration inhibition tests was demonstrated in the H-2 system. The capacity of immune lymphocytes for the MIF production was revealed under their incubation with splenic cells of the congenic or recombinant strains of mice sharing particular private or public H-2 specificities with the donor strains. Selective removal of cytotoxic fraction of lymphocytes resulting from their absorption on the corresponding target cells failed to reduce the capacity of the non-adherent cell population for the MIF production. A fraction of the MIF-producing lymphocytes was found to adhere to the target cells and thereafter to be eluated with the cytotoxic lymphocytes. MIF-producing and target-destroying T-lymphocyte populations are supposed to have antigen-binding receptors differing in their affinity and structural arrangement on the cell surface.
The appearance of specific hypersensitivity to virus antigens was examined in mice infected intravenously with vaccinia virus. Both immediate hypersensitivity, transferable by serum, and delayed-type hypersensitivity, transferable only by cells, were apparent 8 days after infection and demonstrable for at least a further 130 days. Production of macrophage migration inhibitory factor by lymphocytes from infected mice was measured directly in terms of inhibition of migration by antigen or indirectly by determining the effect of soluble factors elaborated by the stimulated lymphocytes. The irregular results may have been the resultants of antigen-mediated macrophage stimulation, toxicity and induction of migration inhibitory factor. Transformation of spleen cells - presumably lymphocytes - from infected mice could be induced in vitro by virus antigens for at least 139 days after infection. Virus/lymphocyte interaction appears to be a particularly fruitful area for further study.
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