Production Of Migration Inhibitory Factor Research Articles

Suppression of lymphokine production by macrophages infiltrating murine virus‐induced tumors

Immune spleen cells from mice injected with murine sarcoma virus (MSV) can produce migration inhibition factor (MIF) in vitro after stimulation with intact tumor cells. We have now found that this effector function can be regulated by suppressor macrophages present in the tumor. The cells from the tumor (CfT) exert a strong suppressive effect even after treatment with anti-Thy 1.2 plus complement but lose this capacity if adherent or phagocytic cells are removed, suggesting that the macrophage is the suppressor cell. The effects of the suppressor cells were exerted on a proliferation-independent function of the lymphocytes, since MIF production, as well as its suppression, occurred even after proliferation of the lymphocytes was blocked by mitomycin-C. The implications of these findings are that macrophages can suppress some early events involved in the activation of lymphocytes by antigen and that they do so through a mechanism that does not relate to the proliferation of the cells.

Production of Migration Inhibition Factor by Spleen Cells of Normal Rats upon Culture in vitro with Tumor Cells and Cells Expressing Endogenous Virus

Abstract Spleen cells from normal rats of W/Fu, Fischer, Sprague-Dawley, and BDIX strains produced migration inhibition factor (MIF) upon culture in vitro with puromycin-treated rat cell lines expressing leukemia viruses or endogenous C-type viruses. Brown Norway rats were almost invariably unreactive. The MIF reactivity of most normal rats did not appear to be due to environmental stimuli because germfree rats were also positive. There was no apparent age restriction for expression of MIF activity; spleen cells from 1-week-old rats produced detectable levels of MIF, and this reactivity was also found in the oldest tested rats, at 34 weeks of age. In addition to spleen cells, lymphoid cells from the blood, peritoneal cavity, and lymph nodes, but not thymus, also were able to produce MIF in response to tumor cells. When (C58NT)D tumor cells, which were routinely used as the stimulators of MIF production, were inoculated into W/Fu rats, the characteristics or extent of preexisting reactivity were not altered. However, spleen cells of most rats bearing progressively growing (C58NT)D tumors did not show a MIF response in vitro. MIF production by spleen cells was dependent upon the presence of macrophages. Spleen cells depleted of phagocytic and adherent cells were unable to produce MIF upon incubation by tumor cells; however, the MIF response could be restored by adding allogeneic or syngeneic macrophages to the depleted spleen cells. The helper role of macrophages in the generation of MIF by spleen cells did not appear to involve antigen presentation because direct contact between macrophages and the responder cells was not necessary, and soluble factor(s) released by macrophages could substitute for the macrophage helper function. The effector cells of MIF production were nonadherent, lacked detectable Fc receptors for IgG, and were resistant to lysis by anti-T cell serum plus complement. Because the MIF response was present in apparently normal rats, this appears to be an expression of natural immunity that may be analogous to natural killer cell activity.

Effect of Thymic Humoral Factor on Cellular Immune Functions of Normal Children and of Pediatric Patients with Ataxia Telangiectasia and Down's Syndrome

Cellular immune functions of nine Down's syndrome patients and of nine was Ataxia telangiectasia vs. nine normal children and nine cord bloods, were evaluated using in vitro assays of peripheral blood lymphocytes. The in vitro assays included E rosette formation, antilymphocytic cytotoxicity by an antithymic antiserum and leukocyte migration inhibition factor (LIF) production. The mitogens and antigens used were phytohemagglutinin, purified protein derivative, and monilia antigen. The effect of a thymic hormone (THF) on these parameters was evaluated and it was administered therapeutically to three Down's syndrome patients and to two patients with Ataxia telangiectasia. Most deficient T-cell functions were reversed to normal after incubation of the lymphocytes with THF, or after THF therapeutic administration. In two Down's syndrome cases, the clinical course was not altered by THF administration, while one seemed to benefit from it markedly. One of the Atactic patients recovered from a severe viral infection, while the other died from intractable bronchopneumonia.

Immunity to foot-and-mouth disease virus in guinea pigs: clinical and immune responses

Clinical and immune responses were determined for guinea pigs infected with different doses of foot-and-mouth disease virus (FMDV) type A12, strain 119, administered by different routes. Vesicles developed on the tongue or heel pad 1 day after these areas were intradermally inoculated with FMDV. However, vesicles did not develop on the feet and tongue until 3 to 4 days after the intradermal inoculation of FMDV in the flank skin or after intracardiac or subcutaneous inoculation. Infected guinea pigs developed neutralizing antibody, immediate skin reactivity of the Arthus type (4 h), and delayed skin reactivity. In addition to a delayed skin response, the presence of a cell-mediated immune response to FMDV was shown by the specific production of macrophage migration inhibition factor by peritoneal exudate cells in response to FMDV. Kinetic studies showed that neutralizing antibodies were detected at 3 days postinfection, and Arthus and delayed skin reactivity were detected at 4 days postinfection. Some guinea pigs developed either mild or subclinical infections. Regardless of the dose of infectious virus, the route of inoculation, the severity of disease, or the time of clinical onset of disease, infected guinea pigs developed similar immune responses.

Histamine-induced suppressor factor (HSF): Further studies on the nature of the stimulus and the cell which produces it

Guinea pig lymphocytes are stimulated by histamine to produce a soluble factor with immunosuppressive properties. This factor, termed histamine-induced suppressor factor or HSF, abrogates the production of migration inhibitory factor (MIF) and proliferative response to specific antigen. In the present study we have determined the lymphocyte subpopulation which elaborates HSF, the lymphoid tissue source, the kinetics of its generation in relation to immunization, and the nature of the histamine receptor involved in modification of the release of HSF. HSF activity could be detected in populations of cells from spleen and lymph nodes prior to active immunization of the donor, but not in cells from the donor's blood or thymus. Following immunization with ortho-chloro benzoyl-bovine γ-globulin in complete Freund's adjuvant (CFA), more HSF activity was detected in cells from the donor's spleen and lymph nodes. The peak response was seen 2 weeks postimmunization when significant amounts of HSF also were made by cells from the blood and thymus. Concentrations of T-cell-enriched and B-cell-enriched populations were tested for their ability to make HSF. We found that T-cell-enriched, but not B-cell-enriched populations, made significant amounts of HSF. Cells from the lymph nodes of immunized donors were chromatographed over affinity columns made of insolubilized conjugates of histamine with albumin. The nonretained cells were unable to generate HSF, whereas HSF activity was detected in the cells that were retained by the columns. This finding strongly suggests that the HSF-producing cells have receptors for histamine. Cells from CFA-immune lymph nodes were incubated with H 1 (2-methyl histamine) and H 2 (4-methyl histamine) agonists to determine their relative potency and, therefore, the nature of the histamine receptors on these cells that were modifying HSF release. Although both agonists could induce generation of HSF when high concentrations (10 −3 M) were used, only the H 2 agonist stimulated production or release of HSF at lower concentrations (10 −5 M). These HSF-producing cells appear to be selectively sensitive to H 2 agonists and likely have a predominance of H 2 receptors. Allergic mediators other than histamine were studied to determine their ability to allow elaboration of HSF-like activity from CFA-immune lymph node cells. Serotonin (10 −3 M), slow-reacting substance of anaphylaxis (100 units/ml), eosinophil chemotactic factor (tetrapeptide; 10 −5 M), and prostaglandin E 1 (10 −4 M) were unable to induce HSF-like activity in lymph node cells from donors immunized with CFA. Furthermore, other agents which raise intracellular levels of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) such as isoproterenol and cholera toxin, as well as the dibutyryl form of cyclic AMP itself, were also unable to generate HSF-like activity. Thus, histamine is unique among the allergic mediators in stimulating elaboration of the suppressive substance. These findings also suggest that the ability of histamine to stimulate HSF may not reside in the conventional pathway linked to cAMP accumulation, but rather to an as yet undefined pathway of cell activation. A model is presented which further implicates histamine as a modulator of cellular immune reactions.

SIGNIFICANCE OF MIGRATION STIMULATORY FACTOR IN HUMAN RENAL ALLOTRANSPLANTATION

Seventy-four recipients of related donor renal allografts were tested for the presence of cellular immunity to specific donor lymphocyte antigens using the direct migration inhibition factor (MIF) assay. Responses on the assay fell into one of the following three statistically distinct groups: (1) greater than 20% inhibition of macrophage migration, (2) nonresponsiveness, +/- 10% of control migration, and (3) greater than 12% stimulation of macrophage migration. Migration stimulation was shown to be reproducible and to correlate well with a very benign post-transplant clinical course. The production of migration stimulatory factor appears to be an immunological response analagous to the production of migration inhibition factor.

Immunobiology and species distribution of Mycobacterium tuberculosis antigen 5.

The immunobiology and mycobacterial species distribution of immunoabsorbent affinity chromatography-purified Mycobacterium tuberculosis antigen 5 have been studied. In delayed hypersensitivity skin tests, antigen 5 was nearly equipotent with tuberculin-purified protein derivative in sensitized guinea pigs. In vitro, antigen 5 was capable of stimulating the production of migration inhibitory factor by cultured lymphocytes from sensitized guinea pigs and humans. Antigen 5 stimulated thymidine incorporation by cultured guinea pig lymphocytes but did not stimulate thymidine incorporation by cultured human lymphocytes. Although erythrocytes were readily sensitized with antigen 5 for passive hemagglutination, their use did not offer any advantage over previous hemagglutination techniques for the serodiagnosis or evaluation of patients with tuberculosis. By immunoelectrophoresis and immunodiffusion, antigen 5 was readily identified in culture filtrates of 10 strains of M. tuberculosis and M. bovis but not in those of 30 strains of 12 other myobacterial species.

Leukocyte Migration Inhibition Factor in Atopic Dermatitis: Induction by Concanavalin A in Vitro

Assays for the production of leukocyte migration inhibition factor (LMIF) activity by cultured lymphocytes in the presence of the T-cell mitogen concanavalin A (ConA) have been shown to be highly sensitive for detecting decreases in cell-mediated immunity in patients with malignant diseases and primary and secondary immunodeficiency diseases. We have applied the leukocyte migration inhibition test in agarose using supernatants from ConA-stimulated lymphocyte cultures (indirect leukocyte migration inhibition test in agarose for studying patients with atopic dermatitis. Only 5 of 14 lymphocyte cultures from atopic dermatitis patients produced LMIF when stimulated with ConA, as compared with 34 of 34 controls. This difference is highly significant.

Effect of Chloramphenicol on III Vitro Function of Lymphocytes

The effect of chloramphenicol on the in vitro function of human peripheral blood lymphocytes was studied in assays of lymphocyte transformation and lymphokine production. When lymphocytes were stimulated by phytohemagglutinin, concanavalin A, or pokeweed mitogen in the presence of various concentrations of chloramphenicol, only minimal effects on blastogenesis were noted. However, suppression by chloramphenicol of blastogenesis induced by candida antigen or streptokinase-streptodornase was greater in magnitude and was dose-dependent; blastogenesis was suppressed to 25%--30% or normal levels by concentrations of chloramphenicol of 25--50 microgram/ml. Chloramphenicol had little effect on the production of the lymphokine leukocyte migration inhibition factor by lymphocytes stimulated either by candida antigen or by concanavalin A, whereas puromycin at a concentration of 5 microgram/ml significantly suppressed this response. Thus chloramphenicol appears to suppress antigen-induced lymphocyte blastogenesis significantly but not lymphokine production by stimulated lymphocytes.

Immunologic Features of Chronic Granulomatous Mucocutaneous Candidiasis Before and After Treatment With Transfer Factor

We report the acquisition of skin test sensitivity to Candida albicans antigen and the ability to produce leukocyte migration inhibition factor (MIF) by a Candida-negative patient with chronic granulomatous mucocutaneous candidiasis after treatment with dialyzable transfer factor (TFd). The TFd was acquired from Candida-positive healthy donors. Three of seven attempts to transfer Candida skin test sensitivity were successful, and the acquired skin reactivity lasted for 12 to 21 days. The acquisition of cellular immunity to Candida was demonstrated in vitro by production of leukocyte MIF. No Candida-induced lymphocyte transformation was observed before or after TFd injection. The TFd did not cause Candida-induced blast transformation when added directly to cultures of lymphocytes from the patient. Pain, tenderness, redness, and edema were observed around the Candida granulomas on each occasion when the skin test to Candida became positive. Two weeks after TDd injection, the proliferative response of peripheral blood lymphocytes increased, as measured by incorporation of tritiated thymidine into lymphocytes within the first hour of in vitro incubation.

Evidence for cell-mediated autoimmunity in patients with pemphigus vulgaris and bullous pemphigoid

Lymphoid cells from 4 of 5 patients diagnosed as pemphigus vulgaris (PV) and 6 of 7 patients diagnosed as bullous pemphigoid (BP) demonstrated specific cell-mediated immunity by the production of migration inhibitory factor (MIF) in the presence of autologous epidermal saline extracts. Clinical treatment of these patients with immunosuppressive agents resulted in a state of unresponsiveness of their lymphoid cells to similar concentrations of the antigen. Controls consisted of lymphoid cells from patients with bullous burns or various drug allergies which failed to show significant MIF production in the presence of autologous skin extract. These studies suggest that both PV and BP patients posses cell-mediated immunity (CMI) to their own autologous tissue antigens and this CMI may play a role in the pathogenesis of these diseases.

Antigen and Mitogen Induced Production of Macrophage Migration Inhibitory Factor in the Mouse

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.

Cell‐mediated immunity to myelin basic protein in Lewis rats made unresponsive to experimental allergic encephalomyelitis

Lewis rats with experimental allergic encephalomyelitis (EAE) exhibited cell-mediated immunity to myelin basic protein as determined both with in vivo and in vitro assays. Positive skin test reactions and production of migration inhibitory factor (MIF) were observed before onset and after recovery from EAE. Rats rendered unresponsive to EAE exhibited in vitro cell-mediated immunity to basic protein, although in vivo manifestations were depressed. However, tolerant rats failed to respond to the encephalitogenic determinant; rats with EAE exhibited cell-mediated immunity to this region of the molecule. The results indicate that EAE-unresponsive rats possess lymphocytes capable of responding to basic protein, but that reactivity to the encephalitogenic peptide is suppressed.

Immunological memory in tuberculosis: 2. Mediators of protective immunity, delayed hypersensitivity and macrophage migration inhibition in central lymph

Inbred rats were immunized with living BCG and their thoracic duct lymphocytes (TDL) were examined for their ability to mediate tuberculin delayed-type hyper-sensitivity (DTH), antituberculosis protective immunity and migration inhibitory factor (MIF) production. TDL collected 9 days after immunization conferred DTH and protective immunity on normal recipients and were reactive in MIF assays. These MIF-producing TDL were relatively large in size as determined by velocity sedi-mentation. TDL collected 28 days or longer after immunizatin conferred adoptive immunity and produced MIF, but could not transfer DTH. The protective lymphocytes were relatively heterogeneous in size but the most enriched population had a sedimentation velocity of 3.9 mm/h. The MIF-producing TDL segregated with the protective lymphocytes after velocity sedimentation and their size corresponded to that of T-lymphocytes. Although mediators of DTH were absent from “late” TDL, recipients of these cells developed DTH at an accelerated rate after challenge with Mycobacterium tuberculosis . The data suggest that protective and MIF-producing cells belong to the recirculating pool of small lymphocytes whereas DTH is mediated by nonrecirculating lymphocytes.

Leukocyte chemotactic activity in cultures of unstimulated human lymphocytes.

We have shown earlier that unstimulated human lymphocytes in in vitro cultures produce migration inhibitory factor into the supernatant. The evidence of spontaneous lymphokine synthesis is strengthened further by this study, which demonstrates leukocyte chemotactic activity in these culture supernatants. The factor has a molecular weight of more than 5000 daltons, it resisted heating for 15 min at 100 degrees C, and showed maximum activity at dilution 1:4-1:8 of the supernatants.

Cell-mediated immunity to acetaldehyde in alcoholic liver disease demonstrated by leukocyte migration test

To determine whether a sensitization to ethanol metabolites occurs in alcoholic liver disease, reactivity of lymphocytes to nontoxic amounts of acetaldehyde was studied by direct elaboration of migration inhibitory factor (MIF) production. Eighteen alcoholics with various degrees of biopsy-proven liver damage showed increased MIF production in response to acetaldehyde; the mean value of the group differed significantly from 15 healthy controls, 15 subjects with nonalcoholic liver disease, and 15 alcoholics without liver involvement (P less than 0.001, P less than 0.001, P less than 0.02, respectively). Among the alcoholics with liver disease, none individuals (50%) with histological signs of advanced alcoholic hepatitis showed the highest percentage of inhibition of migration; the value differed significantly from the remaining patients with lesser degrees of hyaline necrosis in liver biopsies (P less than 0.005). These results indicate that acetaldehyde is involved in the pathogenesis of alcoholic hepatitis. Clinically, this test might facilitate the selection of patients with alcoholic hyaline necrosis.

Cellular hypersensitivity to autologous tumor extract in patients with breast carcinoma.

The indirect macrophage migration inhibition technique was used to study cellular hypersensitivity to autologous tumor extract in relation to the progress and prognosis of breast carcinoma. Cellular immune response, evidenced by production of a macrophage migration inhibitory factor (MIF), was noted preoperatively in 21 of 63 patients (33 per cent). This reactivity was used at the time of surgery to determine the grade of the primary tumor, lymph node involvement and the stage of the disease according to the TNM system. The five-year survival rate was 76 per cent for patients whose lymphocytes responded preoperatively and 54 per cent for patients whose lymphocytes did not respond, indicating that this assay may be valuable in detecting cellular immune response to breast carcinoma and in evaluating the immunological status of patients.

Immune interferon II. Different cellular site for the production of murine macrophage migration inhibitory factor and interferon

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.

CELLULAR IMMUNITY TO CYTOMEGALOVIRUS IN A PATIENT FOLLOWING BONE MARROW TRANSPLANTATION

Cell-mediated immunity to cytomegalovirus (CMV) was studied in a bone marrow transplant patient with evidence of active CMV infection. The lymphocytes from this patient were found to specifically recognize and respond in vitro by transformation to CMV-infected Wistar-38 fibroblasts and by production of macrophage migration inhibition factor to CMV antigen. In addition, plasma and spinal fluid from the patient were found to contain blocking factor that specifically inhibited the lymphocyte response in the above assays. Biochemical, biophysical, and immunological studies indicate that the blocking factor may be an antigen-antibody complex.

Strain Variations in Murine MIF Production

Abstract Stimulation of murine lymphocytes with antigen or mitogen in vitro can lead to the production of macrophage migration inhibition factor (MIF). In this study, variations in MIF production were examined in various inbred strains of mice. When BALB/c (H-2d) and AKR (H-2k) splenic lymphocytes were cultured with concanavalin A (Con A) in serum-free medium, good MIF production resulted within 24 to 48 hr. C3H/He (H-2k) cells cultured under identical conditions produced low levels of MIF and DBA-2 (H-2d) and C57BL/6 (H-2b) cells made no MIF response. Interestingly, however, C57BL/6 cells could make a good MIF response when the cell cultures were supplemented with 2% fetal calf serum (FCS) or if FCS was added to the supernatants after incubation. A similar pattern was observed with the cultures stimulated with specific antigen. This pattern of reactivity demonstrates that variation in MIF production is not directly related to differences in the H-2 complex. The ability of preformed MIF from one strain to react with target cells from different strains was also examined. It was found that MIF produced by Con A-stimulated lymphocytes of a given strain was capable of inhibiting the migration of allogeneic macrophages as effectively as macrophages from the MIF producing strain. Furthermore, immunoadsorption of MIF-active supernatants with alloantisera directed against H-2 regions failed to remove MIF activity. Taken together, these results suggest that MIF activity as well as production are not under direct H-2 control and that the MIF molecule does not share antigenic determinants coded by the murine major histocompatibility complex.