Mastocytoma cell migration in vitro: inhibition by MIF-containing supernatants.

Supernatants with macrophage migration inhibition factor (MIF) activity were obtained from cultures of antigen-stimulated guinea pig and human lymphocytes, and from SV40-infected monkey kidney cells. The monkey and human but not guinea pig preparations were effective in inhibiting migration of mastocytoma cells as well as macrophages. This inhibition of migration was not associated with cytotoxicity and was reversible.     

The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs

The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma₁ + gamma₂ globulins, gamma₁ globulins, gamma₂ globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen.     

Identification of the principal cell type yielding immune RNA capable of transferring tumor-specific cellular cytotoxicity

A search was made for the lymphoid cell type(s) which are the source of immune RNA (I-RNA) capable of transferring tumor-specific cell-mediated cytotoxicity (CMC). Hartley guinea pigs were immunized with syngeneic murine fibrosarcomas (BP-10 or BP-11) induced by 3,4-benzo(a)pyrene in C3H/HeJ mice, and the I-RNA was extracted individually from their spleens, lymph nodes, and peritoneal exudate (PE) cells. All three I-RNA preparations were able to convert normal C3H/HeJ mouse lymphocytes to effector cells significantly cytolytic to the specific syngeneic mouse tumor in vitro. Furthermore, lymphocytes and macrophages were purified from the spleens, lymph nodes, and PE cells of tumor-immunized guinea pigs. I-RNA was extracted from these purified cell populations and also from the pooled guinea pig lymphoid tissues. Normal C3H/HeJ lymphocytes were incubated with each type of I-RNA and tested in vitro for CMC against the specific tumor cells. Significant CMC against BP-10 targets was observed with mouse lymphocytes incubated with I-RNA extracted from pooled lymphoid tissues of BP-10 tumor-immunized guinea pigs. There was a reduced but still significant CMC when mouse lymphocytes were incubated with I-RNA extracted from purified guinea pig lymphocytes, whereas there was a markedly increased CMC when the I-RNA was extracted from purified guinea pig macrophages. As indicated by sucrose density gradient analysis, the lesser effectiveness of lymphocyte I-RNA was not due to RNA degradation resulting from lymphocyte purification or I-RNA extraction. Treatment of all types of I-RNA with RNase abrogated the transfer of CMC, whereas treatment of I-RNA with DNase or pronase did not. RNA extracted from the lymphoid tissues of guinea pigs immunized with complete Freund's adjuvant without tumor was ineffective. Mouse lymphocytes incubated with BP-10 macrophage I-RNA destroyed BP-10 but not BP-11 tumor cells, whereas lymphocytes incubated with BP-11 macrophage I-RNA killed BP-11 but not BP-10 tumor cells, thus indicating tumor specificity of the immunity transferred by macrophage I-RNA. Our results suggest that macrophages are the principal source of I-RNA capable of transferring tumor-specific CMC.     

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.     

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.     

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.     

Antigen handling by guinea pig macrophages: further evidence for the sequestration of antigen relevant for activation of primed T lymphocytes.

Guinea pig macrophages can take up sufficient 2,4 dinitrophenyl guinea pig albumin during a brief in vitro exposure at 37 degrees C to trigger proliferation and lymphokine production with primed T lymphocytes on subsequent co-culture. Treatment of such antigen-bearing macrophages with trypsin, a procedure which removes surface antigen, does not alter the ability of such macrophage to initiate the release of migration inhibition factor from sensitized T lymphocytes. In addition, formation of antigen-specific rosettes between primed T cells and antigen-bearing macrophages is not blocked by high concentrations of antibody directed against the antigen mediating this interaction. Similarly, primed T lymphocyte DNA synthesis induced by antigen-bearing macrophages is not inhibited by specific antibody to that antigen. These data support the conclusion that the fraction of macrophage-associated antigen which is relevant to T lymphocyte activation does not reside on the macrophage surface but rather remains in a restricted compartment from which it is accessible to the T cell but unavailable to either blockade by specific antibody or removal by proteolytic enzymes.     

In vitro correlates of hapten-specific delayed hypersensitivity

Azobenzenearsonate conjugates of l and d-amino acid polymers were studied in respect to their ability to stimulate lymphocytes from guinea pigs sensitized to the hapten. Using the two in vitro techniques of thymidine incorporation and inhibition of macrophage migration, it was shown that only conjugates of the l-amino acid polymers were active.The results confirm previous in vivo studies and suggest as one possibility that a preliminary processing event by macrophages may be obligatory for all associated phenomena of hapten-specific delayed hypersensitivity.     

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.     

Characterization of guinea pig macrophages: I. Mobility and maturation of peritoneal cells following inflammatory stimuli

The wide spectrum of activities of macrophages may, in part, be a reflection of their different subpopulations. Sedimentation velocity was used in separate different guinea pig macrophage populations which were then compared in assays which measure some of the parameters of inflammatory responses. Normal resident, thioglycollate-induced exudates or cells elicited by ip injection of tuberculin into BCG-sensitized animals were studied. In thioglycollate exudates the small, possibly recently derived monocytes were most responsive to chemotactic agents whereas populations with high sedimentation velocity were more phagocytic, responsive to MIF and produced more LAF. By contrast, all macrophage populations or subpopulations from sensitized animals challenged with PPD were unresponsive to lymphocyte-derived chemotactic factor, endotoxin-activated serum, or to N-formyl-l-methionyl-l-phenylalanine in chemotaxis assays. In addition, macrophages from these animals did not migrate from capillary tubes in MIF assays. The lack of migration is presumably due to the action of MIF on these cells in vivo. Cells with low sedimentation velocity (predominantly lymphocytes), however, did migrate from capillary tubes. We conclude that small cells elicited by a nonspecific stimulus are probably attracted to the inflammatory site by chemotaxins and must subsequently enlarge or mature before they are able to respond to MIF, to be activated to produce LAF, or to exhibit extensive phagocytosis.     

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.     

Macrophage-T cell interaction mediated by immunogenic and non-immunogenic forms of a monofunctional antigen

Summary As an approach to the elucidation of the essential steps in the immune pathway, the uptake and retention of immunogenic and non-immunogenic analogs of a monofunctional antigen by guinea pig macrophages and the efficiency of macrophages pulsed with the compounds to present antigen to sensitized T lymphocytes were compared. L-Tyrosine-azobenzene-p-arsonate (RAT) and its non-immunogenic analog, 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate (RAN), react similarly with antiarsonate antibody, but RAN, unlike RAT, is unable to induce cellular immunity in guinea pigs. The uptake and retention patterns of the two compounds by macrophages differed in that, at a given time, more RAN than RAT was retained and detectable on cell surfaces by anti-arsonate antibody. Equivalent numbers of T lymphocytes from guinea pigs sensitized to RAT formed antigen-dependent clusters with macrophages pulsed with either RAT or RAN after 24 hr in culture, but not with macrophages pulsed with an azobenzenoid compound of unrelated specificity. On the other hand, T lymphocytes from guinea pigs immunized with RAN showed no significant capacity to bind to macrophages which had been pulsed with any of the compounds. The number of lymphocytes from RAT-sensitized animals which bound to RAT-pulsed macrophages remained relatively stable over a 48 hr period, whereas clusters of the same lymphocytes with RAN-pulsed macrophages dissocitated to background levels within that time. Early cluster formation mediated by RAN, as well as its ability to induce transient specific T cell unresponsiveness to RAT in vivo, indicate that T cells are capable of recognizing (binding) the non-immunogen. However, such early, and perhaps weak, interaction with RAN-pulsed macrophages did not induce DNA synthesis by T cells. Anti-Ia serum completely blocked cluster formation mediated by either RAT or RAN. Thus, the only significant distinction disclosed by these studies between the immunogenic and non-immunogenic compounds was the stability of macrophage-T cell interaction as determined by the persistence of antigen mediated cell clusters in culture, suggesting that this may be a factor in immunogenic discrimination.Abbreviations ABA azobenzenearsonate - BSA bovine serum albumin - CFA complete Freund's adjuvant - IFA incomplete Freund's adjuvant - KLH keyhole limpet hemocyanin - LNC Lymph node cells - MHC major histocompatibility complex - PEC peritoneal exudate cells - PEL peritoneal exudate lymphocytes - RAN 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate - RAT L-tyrosine-azobenzene-p-arsonate - TAT L-tyrosine-azobenzene-p-trimethylammonium chloride Aided by USPHS Grant AI 05664.     

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.     

Modification of the agarose drop micromethod for studying mouse macrophage migration inhibition factor

A modification of the drop agarose micromethod for the study of the factor inhibiting the migration of mouse macrophages is proposed. The syngeneic system was used instead of type RPMI-1640 complex culture media and calf embryonal serum, commonly used in the work with mouse macrophages. The production of the factor inhibiting the migration of macrophages by sensitized lymphocytes from mice of different strains has been shown to occur in vitro under the influence of pertussis corpuscular antigen. The method of obtaining supernatant containing macrophage migration inhibiting factor, from immune mouse lymphocyte culture is proposed. This supernatant may be used for, the analysis of the above-mentioned factor and other lymphokins.     

Further characterization of the putative glycolipid receptor for mif: Role of fucose associated with an acidic glycolipid

Water soluble glycolipids were extracted from guinea pig macrophages. These glycolipids, when incubated with macrophages, augment the cells' response to migration inhibitory factor. The glycolipids were fractionated by diethylaminoethyl-Sephadex ion exchange chromatography into neutral and acidic fractions. Only the acidic glycolipid fraction was able to enhance the responsiveness of macrophages to migration inhibitory factor. Additional studies indicate that the enhancing activity of these glycolipid preparations can be abrogated by the removal of terminal fucose residues with α-L-fucosidase. The possibility that fucose functions as an essential component of a macrophage glycolipid receptor for migration inhibitory factor is discussed.     

Composition of splenic T lymphocytes in allophenic mice.

Guinea pig peritoneal macrophages were activated in vitro by culturing with MAF (macrophage activating factor)-containing fractions from stimulated lymphocytes. These macrophage preparations demonstrate a 60% increase in the production of prostaglandins of the E series (PGE) when compared with macrophages cultured with fractions from unstimulated lymphocytes. PGE accumulation in macrophage cultures is maximal after 24 hr with MAF; tumor cytotoxicity is also maximal at this time. The final PGE concentration in cultures of activated macrophages averaged 3 × 10^?8M.     

Strain differences in susceptibility to experimental allergic encephalomyelitis and the immune response to the encephalitogenic determinant in inbred guinea pigs.

Strain differences in susceptibility to experimental allergic encephalomyelitis (EAE) in guinea pigs were correlated with the cellular immune response to the basic encephalitogenic protein (BE). The response to BE was determined in strains 2 and 13 guinea pigs in vivo by the delayed hypersensitivity skin test and in vitro by the lymphocyte transformation technique. The response to the intact BE of both heterologous (bovine) and homologous (guinea pig) origins was indistinguishable between the two strains. Guinea pigs sensitized with the guinea pig BE showed complete cross-reaction when tested with the bovine BE. On the other hand, there appears to be significant differences in the response to specific determinants on the molecule. Thus, only strain 13 and F₁ hybrids which are susceptible to EAE responded to the encephalitogenic nonapeptide (residue 114–122 of the BE molecule), whereas strain 2 guinea pigs which are resistant to EAE did not respond to this determinant.     

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.     

Possible role of macrophage glycolipids as receptors for migration inhibitory factor (MIF).

Guinea pig peritoneal exudate cells incubated with water soluble glycolipids obtained from macrophages show an enhanced response to migration inhibitory factor. Incorporation of these glycolipids into liposomes greatly facilitates their interaction with indicator cells. Enhancement of peritoneal exudate cell responsiveness to migration inhibitory factor was specific for glycolipids from guinea pig macrophages. Glycolipids extracted from guinea pig brain and polymorphonuclear leukocytes as well as several bovine and porcine glycolipids had no effect. Specificity of enhancement was not due merely to a preferential association of macrophage glycolipids with indicator cells. The possible role of macrophage glycolipids as receptors for MIF 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.