Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

In vitro studies on transplantation immunity. I. MIF production by sensitive lymphocytes in mice

Sensitized lymphocytes from mice immunized with skin homografts produce migration inhibitory factor upon incubation with lymphocytes (antigen) from the sensitizing strain. The MIF is produced within 14 hr following incubation of sensitized lymphocytes and antigen. In this reaction, antigenic specificity is a prerequisite for MIF production; however, the action of MIF transcends the strain barrier. Also, MIF produced in homograft reactions in mice inhibited the migration of peritoneal cells from normal guinea pigs. Finally, lymphocytes from mice bearing skin homografts do not develop the capacity to produce MIF prior to the rejection of the sensitizing skin grafts.     

Electrophoretic and immunoelectrophoretic studies of sera from normal, tuberculous, and noninfected tuberculin-sensitive guinea pigs

Dardas, T. J. (Michigan State University, East Lansing), and V. H. Mallmann. Electrophoretic and immunoelectrophoretic studies of sera from normal, tuberculous, and noninfected tuberculin-sensitive guinea pigs. J. Bacteriol. 92:76-81. 1966.-Normal guinea pig serum was separated into seven fractions by electrophoresis on cellulose acetate membranes. Thirty antigens were found by immunoelectrophoresis: albumin, 6 alpha(1) globulins, 11 alpha(2) globulins, 6 beta(1) globulins, 5 beta(2) globulins, and gamma globulin. Hyper-alpha(2)-globulinemia was detected in sera from guinea pigs 14 days after inoculation with viable virulent Mycobacterium bovis. An additional alpha(2) globulin, not demonstrable prior to infection, was detected concomitantly with the hyper-alpha(2)-globulinemia by immunoelectrophoresis. The additional alpha(2) globulin was tentatively named alpha(2)-T. It persisted until the death of the guinea pigs. Neither hyper-alpha-globulinemia nor the alpha(2)-T antigen was detected by cellulose acetate electrophoresis and immunoelectrophoresis of sera from guinea pigs sensitized with heat-killed M. bovis. Both changes were due to the disease, not to delayed sensitivity alone.     

Effect of C-reactive protein on peritoneal macrophages. I. Human C-reactive protein inhibits migration of guinea pig peritoneal macrophages

The effect of human C-reactive protein (CRP) isolated and purified from pooled patients' sera on macrophage function, especially on macrophage migration, was studied. Peritoneal exudate cells (PEC) from guinea pigs were used for macrophage migration inhibition (MMI) test of capillary method. Migration of either PEC or adherent purified macrophages exposed to CRP were inhibited dose-dependently. These findings indicate that CRP inhibits macrophage migration directly, not via activation of lymphocytes contained in PEC. As control, we examined the effect of normal human serum, anti C-polysaccharide antibodies isolated from patients' sera, and free endotoxin at the dose contaminated in CRP preparation on macrophage migration and found that none of them were effective. The effect of CRP on MMI of sensitized PEC exposed to antigen was also studied. Large amounts of CRP inhibited MMI induced by antigen, indicating the possibility that CRP may act on macrophages competitively with migration inhibitory factor (MIF) and may modulate MMI. CRP possesses MIF-like activity and may play a functional role at the site of tissue injury by causing the accumulation of macrophages.     

Production and in vivo effect of antibodies against guinea pig lymphokines

Supernatants from guinea pig lymph node lymphocytes stimulated with insoluble Concanavalin A in serum-free medium were fractionated by Sephadex chromatography and preparative electrophoresis. The isolated fraction possessed migration inhibition, mitogenic, and skin reactive activities. Associated with these were apparently two newly synthesized haemoproteins of unknown function. Antibodies were prepared against this partially purified lymphokine fraction. MIF produced by sensitized lymphocytes activated with an antigen (PPD tuberculin) could be completely absorbed from whole supernatants by immunoadsorbent columns prepared with that antibody whereas mitogenic factor and skin reactive factor were not retained. The anti-lymphokine antiserum totally inhibited the delayed skin response of sensitized guinea pigs challenged with PPD.     

Staphylococcal enterotoxin B induced release of macrophage migration inhibition factor from normal lymphocytes

Staphylococcal enterotoxin B (SEB), a potent lymphocyte mitogen, inhibits migration of peritoneal exudate cells from most guinea pigs but does not inhibit migration of purified macrophages. Experiments were designed to test the ability of highly purified SEB to induce normal lymphocytes to release migration inhibition factor (MIF). Supernatants of lymph node lymphocytes cultured with SEB inhibited the migration of purified macrophages, indicating the release of a migration inhibition factor. Mitomycin-C blocked the SEB-induced release of MIF. SEB-induced MIF localized in the albumin fraction on Sephadex G-200 chromatography. Antibody to SEB specifically blocked the inhibitory effect of SEB on migration of normal guinea pig peritoneal exudate cells.     

Release of macrophage migration inhibitory factor(s) from lymphocytes stimulated by streptococcal preparations

Lymphocytes from apparently healthy subjects, incubated for 5 hours with cellular components or extracellular products of group A streptococci and then washed and reincubated, were found to release factor(s) capable of inhibiting guinea pig lung macrophage migration (“indirect method”). Inhibitition of macrophage migration was also obtained when the same preparations were tested directly on guinea pig lung cells, a macrophage-lymphocyte population (“direct method”). The guinea pigs had not been experimentally sensitized. The inhibition of migration appeared to depend on the presence of lymphocytes among the macrophages, since macrophages purified by repeatedly discarding nonadherent cells proved resistant to the migration inhibiting activity of the most active Streptococcal preparation, a 20 × concentrated filtrate. Reconstitution of the original lymphocyte-macrophage mixture reestablished the reactivity. The macrophage migration inhibition did not correlate with the age of the guinea pigs. It could not be obtained with preparations of group D streptococci or of Salmonella paratyphi. Group C streptococci did not inhibit the macrophage migration with the indirect method, but it did with the direct one.The factor(s) released into the medium on stimulation of apparently normal lymphocytes by Streptococcal preparations was relatively heat resistant, nondialyzable, and DNase and RNase resistant; its release was inhibited by puromycin. Pretreatment of the cells with trypsin prevented the absorption of the factor(s) and left migration unaffected. These characteristics are similar to those previously described for the migration inhibitory factor (MIF) produced by the interaction of sensitized lymphocytes and specific antigens. Whether or not these similarities indicate an identity remains to be determined.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. III. A soluble factor released from LPS-stimulated peritoneal adherent cells effective in antigenic activation of sensitized lymphocytes.

Antigen-induced production of migration inhibitory factor (MIF) by sensitized lymphocytes requires macrophages to effectively stimulate lymphocytes with soluble antigen in vitro. The present study showed that macrophage-depleted lymphocytes of sensitized guinea pigs could be activated with antigens when the culture supernatant of peritoneal adherent cells pulse-stimulated with a macromolecular fraction of bacterial lipopolysaccharide (LPS) was added to the lymphocyte culture. The apparent macrophage-replacing activity was found in the fraction which emerged slightly ahead of serum albumin upon gel filtration of the culture supernatant, and the activity was shown to be destroyed by heating at 65 °C for 30 min or by trypsin digestion. These results appeared to show that the activity was due to a protein component, most probably released from macrophages. Two-step culture experiments revealed that the soluble factor should be present in the early stage of the culture to activate the macrophage-depleted immune lymphocytes with antigen, as well as in the later stage when the presence of antigen in the medium is no longer required. Furthermore, the factor was shown to act in the activation of a T-cell-enriched fraction of immune lymphocytes. The factor appeared to be playing some essential role in making an antigenic stimulus effective for the activation of immune lymphocytes.     

Antibodies to guinea pig lymphokines. II. Suppression of delayed hypersensitivity reactions by a "second generation" goat antibody against guinea pig lymphokines.

A "second generation" antibody to a highly purified lymphocyte product was raised in a goat against material eluted from a rabbit anti-guinea pig lymphokine immunoadsorbent column. This anti-lymphokine serum, in constrast to anti-lymphocyte serum (ALS) did not appear to contain cytotoxic antibodies directed against membrane antigens on guinea pig lymph node lymphocytes. Furthermore, the anti-lymphokine serum did not inhibit the formation of spontaneous T rosettes nor significantly depress lymphocyte response to mitogens. The anti-lymphokine serum totally suppressed the delayed skin reactivity to PPD and contact sensitivity to DNCB when injected intradermally around the site of antigen challenge. By contrast, intradermally injected ALS did not appear to suppress the PPD response in sensitized guinea pigs. Intravenously and i.p. administered anti-lymphokine serum was somewhat less effective in suppressing the delayed skin response to PPD. The intradermal injection of the antiserum had no effect on nonspecific inflammation evoked by turpentine-olive oil or on the extravasation of circulating Evans blue evoked by intradermally injected histamine. Histologic examination of 24-hr DNCB-induced skin lesions from sensitized guinea pigs treated with intradermally injected anti-lymphokine serum showed marked reduction of mononuclear infiltration of the dermis and of epidermal lesions, as compared with skin sites taken from sensitized animals pretreated with normal goat serum. The anti-lymphokine serum injected i.v. also markedly reduced the perivascular infiltration of the dermis and subcutis in skin reaction sites from sensitized animals challenged with PPD. Intravenous treatment with ALS for 3 consecutive days caused extensive depletion of the paracortical areas of peripheral lymph nodes whereas treatment with normal serum and anti-lymphokine serum caused no such depletion. It is proposed that the anti-lymphokine serum is directed against activated lymphocyte products, one of them being MIF. These products are involved in the mediation of delayed hypersensitivity reactions. This is in marked contrast to ALS, the suppressive action of which appears to be central rather than peripheral.     

Guinea pig macrophage agglutination factor is antigenically distinct from migration inhibition factor and immunoglobulin.

Lymph node cells from guinea pigs with specific delayed hypersensitivity release macrophage agglutination (MAggF) and migration inhibition factors (MIF) upon exposure to antigen or concanavalin A in serum-free medium. MAggF in culture supernatants was absorbed neither by immunoabsorbents made with a rabbit anti-guinea pig lymphokine serum that removed MIF, nor by immunoabsorbents made with rabbit anti-guinea pig Ig. These results suggest that MAggF is antigenically distinct from MIF and Ig.     

Histamine-induced suppressor factor (HSF): effect on migration inhibitory factor (MIF) production and proliferation.

Histamine added in vitro to cultures of sensitized lymphocytes suppresses antigen-induced production of migration inhibitory factor (MIF) and proliferation by these cells. Recent studies have suggested that lymphocytes bearing histamine type-2 receptors play a regulatory role in these in vitro responses. The present studies were undertaken to determine if suppressor function by cells having histamine receptors was mediated through a soluble product. It was found that lymph node cells from nonimmune or immune strain 2 guinea pigs elaborate a nondialyzable factor into the culture supernatant when incubated with 10(-3) to 10(-5) M histamine (histamine-induced suppressor factor of HSF). HSF, when cocultured with sensitized lymphocytes, suppressed their MIF and proliferative responses to antigen. HSF was made by lymphocytes but not macrophages. Its production could be blocked by an H2 receptor antagonist (burimamide) but not an H1 receptor antagonist (chlorpheniramine). Furthermore, the inhibitory effect of HSF was reversible as lymphocytes washed free of the factor after 24 hr and recultured with fresh medium and antigen were able to produce MIF. Gel filtration by Sephadex G-100 chromatography indicated that HSF had an approximate m.w. of 23,000 to 40,000. These results suggest that the release of histamine at the sites of immediate hypersensitivity reactions, possibly by generating HSF activity, may play a regulatory role in the subsequent development of cellular-immune reactions at the same site.     

Properties of TAS-1D3, a Tuberculin-Active Substance from BCG,in Regard to Delayed Hypersensitivity

The properties of TAS-1D3, a tuberculin-active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS-1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS-1D3 were similar to those of the reaction to PPD. Moreover, TAS-1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS-1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat-killed M. tuberculosis Aoyama B or heat-killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity: I. Suppression of cutaneous delayed-type hypersensitivity by migration inhibitory factor

The macrophage migration inhibitory factor (MIF) fraction was prepared from the immunoadsorbent column by using anti-guinea pig MIF antiserum. Suppression of cutaneous delayedtype hypersensitivity was achieved by intraperitoneal injection of the MIF fraction into the animals bearing macrophage-rich peritoneal exudates. Skin reactions induced by phytohemagglutinin (PHA) were also suppressed in these animals. Reactivity to skin reactive factor (SRF) was suppressed in these animals as well. The sera obtained from these animals exhibited the inhibitory activity against production of lymphokines from sensitized lymphocytes.     

Cell surface receptors for lymphokines. I. The possible role of glycolipids as receptors for macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF).

Incubation of culture supernatants from concanavalin A-stimulated guinea pig and rat lymphocytes with protein-free preparations of bovine brain gangliosides abolished their macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF) activity. The identity of the MIF/MAF-binding component(s) present in these glycolipid mixtures has yet to be established, but adsorption experiments using purified preparations of mono- (GM₁, GM₂, and GM₃), di- (GD_1a), and trisialogangliosides (GT₁) were negative. Since these gangliosides account for over 90% of the glycolipid content in brain ganglioside mixtures it appears that the MIF-binding component(s) is present only in very small amounts. Treatment of guinea pig peritoneal macrophages with liposomes containing similar brain gangliosides or water-soluble glycolipids extracted from guinea pig macrophages enhanced their responsiveness to MIF. The enhanced response to MIF of liposome-treated macrophages was abolished by incubation of the treated macrophages with fucose-binding lectins (Lotus agglutinin and Ulex europaeus agglutinin I) before exposure to MIF, suggesting that the MIF-binding component donated by the liposomes may be a fucose-containing glycolipid. The possible role of glycolipids as surface receptors for MIF and MAF is discussed.     

In vitro and in vivo studies on the mechanism of experimental autoimmune thyroiditis in guinea pigs

Thyroid explants of inbred strain 13 guinea pigs were grown in a semisynthetic medium containing 0.3 IU of thyroid-stimulating hormone. The monolayer retained the capacity in vitro to form thyroglobulin. Sensitized lymphocytes from animals with autoimmune thyroiditis could specifically lyse these thyroid target cells in vitro in the presence of an appropriate amount of specific antigen. This cytotoxicity was not observed in thyroid epithelial cells which had been incubated (a) with normal lymphocytes or (b) with purified macrophages either from normal animals or from animals with autoimmune thyroiditis. When thyroid cells were incubated with hyperimmune antithyroglobulin serum, cytolysis did not occur, whether or not complement was added. The cytopathic effect of sensitized lymphocytes was further demonstrated to be caused by a soluble cellular product, termed thyroid cytotoxic factor, or TCF, which was released from sensitized lymphocytes under the stimulation of specific antigen, thyroglobulin, and could exert a cytotoxic effect directly on the target cells. Direct cell-to-cell contact was not required in this type of cell-mediated cytolysis.     

Cytotoxicity of sera against brain and spinal cord antigens in relation to lymphocytes of varying origin]

Rabbit sera against antigens prepared from the brain and the spinal cord antigens were investigated in a cytotoxic test with mouse and guinea pig lymphocytes. None of the sera exerted a cytotoxic effect on the bone marrow lymphocytes. The sera against mouse brain and spinal cord and guinea pig brain and myelin isolated from it exerted the greatest cytotoxic activity; the cytotoxicity was maximum against the thymocytes, less pronounced against the lymph node lymphocytes, and least--against the spleen cells. The cytotoxicity of the sera against the bovine spinal cord homogenate, myelin and the basic protein isolated from it was the minimal and equal with lymphocytes from any of the three mentioned sources. The serum against the encephalitogenic polypeptide 2c was practically devoid of the cytotoxic activity. The encephalitogenic activity of the 2c fraction was greater than that of the myelin and basic protein from the bovine spinal cord. Experiment of antibrain serum absorption suggested that the brain cortex contained a cross-reacting antigen. The subcutaneous injection of a relatively high dose (224 x 10(6)) of thymocytes in the complete Freund's adjuvant failed to induce the development of allergic encephalomyelitis in guinea pigs.     

Production of migration inhibitory factor (MIF) by human leukocytes following exposure to Epstein-Barr virus.

The ability of Epstein-Barr virus (EBV) to induce a cell-mediated immune response (CMI) in cultures of human leukocytes was investigated. Partially purified EBV, obtained from culture fluids of AV-1 cells, was inactivated by uv-irradiation. Inactivated virus was mixed with peripheral leukocytes from Hodgkin's disease (HD), infectious mononucleosis (IM) and malignant lymphoma patients as well as from normal individuals in an in vitro culture system. Production of migration inhibitory factor (MIF), as measured by guinea pig macrophage migration inhibition (MMI), was utilized as an indicator of CMI response. Significant differences in MIF response were observed subsequent to exposure of the cells to EBV. Leukocytes from patients in each of the disease categories tested exhibited greater MIF production than did those from the normal controls. There were significant differences in MIF production by leukocytes from the malignant and non-malignant disease categories. Serum from each subject was examined for immunoglobulin specific for EBV capsid antigen (anti-VCA). Although the majority of individuals within the disease categories tested had elevated anti-EBV serum titers, no correlation could be made between elevated anti-VCA titer and levels of MIF production.     

In vitro analysis of immune responses of inbred guinea pigs infected in vivo with Leishmania enriettii and an investigation of active immunization of susceptible animals

Adherent skin cell monolayers have been prepared from the infected area of inbred guinea pigs inoculated with Leishmania enrietti. Cells from those animals most susceptible to disease (2/N) are less able to promote proliferation and the production of macrophage migration inhibition factor (MIF) from leishmania-immune lymphocytes than are infected cells taken from the more resistant 13/N or (2/N × 13/N)F₁ animals. Exposure of immune lymphocytes of all strains to parasite antigen in the relative absence of autologous antigen-presenting cells induced the development of a suppressor pool capable of inhibiting lymphocyte proliferation in response to immunogenic signals. Decreased lymphocyte proliferation during the course of disease may be caused by the endogenous release of products of arachidonic acid metabolism from host monocytes, though along with the decline in DTH reactivity in infected animals, and apparently in concert with the healing of the primary lesion, there occurs an increase in titer of antileishmania antibody in infected animals. Preliminary attempts to confer protection of naive animals by preimmunization with lymphocytes from previously infected, susceptible, or resistant guinea pigs suggested a role for interacting “helper” and suppressor lymphocyte pools in the immunoprotection from leishmania infection.     

Isolation of an antigen fromBlastomyces dermatitidis that is specific for the diagnosis of blastomycosis

The A antigen ofBlastomyces dermatitidis has been isolated and purified by DEAE column chromatography. In the complement-fixation test, the antigen reacted with 10 of 16 sera from patients with proven cases of blastomycosis and was negative with known positive sera from 7 cases of histoplasmosis, 5 cases of coccidioidomycosis, 5 cases of candidiasis, and 5 cases of cryptococcosis. In the enzyme-immunoassay test, 25 of 27 sera from cases of blastomycosis were positive, but all heterologous and normal sera tested were negative. The antigen gave a positive skin test with guinea pigs sensitized with killed yeast-phase cells ofB. dermatitidis and negative skin tests with guinea pigs sensitized with killed yeast-phase cells ofHistoplasma capsulatum.     

Macrophage migration inhibition by complement activators,migration inhibitory factor,and Lotus fucolectin: Evidence for common involvement of cell-associated esterase activity

Activators of the complement pathway were compared with migration inhibitory factor (MIF) and Lotus fucolectin, which mimics MIF, for their macrophage migration inhibitory (MMI) activity. Endotoxin (LPS), cobra venom factor (CVF), and zymosan, known complement (C3) activators, were found to produce dose-dependent MMI activity, similar to MIF, independent of requirements for nonadherent lymphocytes and serum complement. Comparable activity was observed by all migration inhibitors in the presence of freshly harvested unheated or heat-inactivated guinea pig serum as well as zymosan-adsorbed (C3-depleted) serum. Iscove's serum-free medium also promoted migration inhibition confirming a lack of requirement for serum complement in the reaction. Polymyxin B reversed MMI by LPS, but had no effect on the other inhibitors, indicating that the MMI activity of CVF, zymosan, MIF, and Lotus fucolectin was not primarily due to LPS contamination. Peritoneal macrophages (PM), depleted of nonadherent lymphocytes, responded as well as unpurified PM to the complement activators and MIF. ?-Amino-n-caproic acid (EACA), l-lysine, and tranexamic acid (TA), known inhibitors of the fibrinolytic activity of plasminogen activator (PA), were found to reverse migration inhibition of C3 activators, MIF, and Lotus fucolectin. In contrast bovine pancreatic trypsin inhibitor (BPTI) and soybean trypsin inhibitor (STI) had no effect on MMI activity. These results suggest a common mechanism for mediation of migration inhibition by complement activators and MIF which may involve activation of cell-associated complement to produce esterolytic end products capable of triggering the activation process.