Decreased Fc and C3 receptor function in macrophage populations which are refractory to migration inhibitory factor,C3 activators,and immune complex

Guinea pig macrophage populations previously established to be either responsive or refractory to activation by migration inhibitory factor (MIF) and Lotus fucolectin in the macrophage migration inhibition (MMI) assay were further characterized for their MMI response to diverse effectors as correlated with their Fc and C3b receptor function. MIF-refractory populations were found to be uniformly unresponsive to the complement activators: bacterial lipopolysaccharide, cobra venom factor, zymosan, and immune complex. MIF-responsive macrophages were responsive to the same activators. Fc-mediated binding and phagocytosis of IgG-coated sheep erythrocytes (EA) were markedly depressed in freshly harvested refractory macrophages as compared to responsive cells. Fc phagocytosis by refractory populations increased rapidly during 24–28 hr in vitro culture to levels equal to that of responsive cells which corresponded with an increase in their MMI response to MIF. Refractory macrophages also had decreased C3b receptor function as shown by reduced binding and phagocytosis of EAC or serum-coated zymosan and displayed a greater loss in C3b binding capacity than responsive cells during 48 hr in vitro culture. Trypsinization of responsive macrophages rendered them refractory in their MMI response to the various activators and selectively reversed C3b-dependent binding without effect on Fc binding. The plasmin esterase inhibitors, ?-amino-n-caproic acid, tranexamic acid, and l-lysine, previously established to reverse the MMI response to MIF, FBP, and C3 activators were found to inhibit both Fc- and C3-dependent phagocytosis. These results indicate that macrophage populations which are refractory to migration inhibition by MIF and C3 activators also have reduced Fc- and C3b-mediated phagocytic functions as compared to more mature responsive populations.     

Similarities in enhanced glucosamine incorporation by macrophages stimulated with migration inhibitory factor and the fucolectin from Lotus tetragonolobus

Fucose-binding protein (FBP), the fucolectin from Lotus tetragonolobus, was compared with migration inhibitory factor (MIF) for its ability to stimulate [¹⁴C]glucosamine incorporation into trichloroacetic acid (TCA)-insoluble material of guinea pig peritoneal exudate cells (PEC) using a microtiter assay. Both MIF and FBP inhibit macrophage migration and were shown to stimulate glucosamine incorporation in a similar dose response fashion over time. Both unpurified PEC and PEC depleted of nonadherent cells displayed significant levels of glucosamine incorporation when stimulated by MIF or FBP. Tunicamycin and 2-deoxy-d-glucose, known inhibitors of glycosylation, inhibited glucosamine incorporation by control and MIF- or FBP-stimulated PEC. These results confirm the similarities between MIF and FBP in their biological activity for macrophages using a second in vitro correlate of cell-mediated immunity and suggest involvement of enhanced glycoprotein or glycolipid biosynthesis by FBP and lymphokine-activated macrophages.     

Triggering of interferon gamma-primed macrophages by various known complement activators for nonspecific tumor cytotoxicity

Five known complement activators were evaluated for their capacity to directly activate murine macrophages and to trigger activation of lymphokine primed macrophages for nonspecific tumor cytotoxicity. Bacterial lipopolysaccharide (LPS), Lipid A, polyinosinic-polycytidylic acid, cobra venom factor (CVF), and zymosan directly activated macrophages in a dose-dependent fashion at high concentrations. Subactivating concentrations of each of these agents were found to effectively trigger macrophages which were preprimed either by macrophage-activating factor or by murine recombinant interferon gamma for enhanced tumoricidal activity. An Fc receptor blockade with opsonized sheep erythrocytes abrogated LPS-mediated direct activation and triggering of interferon gamma-primed macrophages, but had no inhibitory effect on direct activation or triggering by CVF for nonspecific tumor cytotoxicity. This study characterizes the capacity of a diverse group of known complement activators to serve as second signal triggers for culmination of the activation process of interferon-primed macrophages for nonspecific tumoricidal activity. These findings suggest that complement activators may directly activate macrophages by stimulation of interferon beta production by macrophages for self-priming and, as we have shown, act as self-triggers. The putative role of macrophage-associated complement components in the activation process is discussed.     

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.     

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.     

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.     

Benzisothiazolones as modulators of macrophage migration inhibitory factor

Substituted N-phenylbenzisothiazolones have been investigated as inhibitors of the tautomerase activity of the proinflammatory cytokine MIF (macrophage migration inhibitory factor). Numerous compounds were found to possess antagonist activity in the low micromolar range with the most potent being the 6-hydroxy analog 1w. Compound 1w and the p-cyano analog 1c were also shown to exhibit significant inhibition of the binding of MIF to its transmembrane receptor CD74. Consistently, both compounds were also found to retard the MIF-dependent phosphorylation of ERK1/2 in human synovial fibroblasts.     

Angiotensin II is chemotactic for a T-cell subset which can express migration inhibition factor activity in murine schistosomiasis mansoni

In murine schistosomiasis mansoni, ova induce a delayed-type hypersensitivity, granulomatous response in which angiotensins are produced. Angiotensin II (AII) elicits a chemotaxis for splenic mononuclear cells derived from these infected animals. The effect of AII upon the migration of a T-lymphocyte subset was defined functionally to further delineate this observation. A chemotaxis chamber was developed that permitted collection of large numbers of viable cells which migrate in response to AII. In a direct migration inhibition factor (MIF) assay, MIF activity was demonstrated with 100-fold fewer chemotactically attracted cells as opposed to whole splenic leukocytes. The MIF activity was eliminated by treatment of the cells with anti-Lyt 1.1 or-Thy 1.2 serum and complement. This observation was particularly interesting since migrated and whole spleen cell populations comprised equal numbers of T cells. Incubation of spleen cells with AII prior to assay did not alter MIF activity. These findings suggest that AII is chemotactic for at least one important T-cell subset relevant to the granulomatous response.     

Macrophage migration stimulation factor, migration inhibition factor and the role of suppressor cells in their production.

Guinea pig lymph node lymphocytes and human peripheral blood lymphocytes when stimulated by specific antigen or mitogen will release factors that affect in vitro macrophage migration. Migration inhibition factor production appears to be under the control of suppressor cells which are T lymphocytes. When suppressor cells are generated by stimulation with Con A for 4 days, migration stimulation factor (M.St.F.) activity is found. In other situations where M.St.F. is found this is thought to be due to increased suppressor cell activity. For example, young adults produce this lymphokine when stimulated with Con A, whereas aged individuals produce MIF. Concanavalin A appears to be the mitogen of choice for M.St.F. production, and phytohemagglutinin for MIF production. The release of this putative factor M.St.F. from suppressor T cells helps to explain some of the difficulties that have existed in studies of macrophage migration inhibition.     

Characteristics of T-cells synthesizing macrophage migration inhibition factor in an H-2 system]

Migration inhibition factor (MIF) was found to be produced by T-cells rather than B-cells as shown by the indirect macrophage migration inhibition test in the H-2 system performed with fractionated lymphocytes. MIF-producers appeared to be more sensitive than T-killer cells to the cytotoxic action of anti-theta-antibodies, plus complement. MIF synthesis by lymph node cells developed earlier after immunization than the cytotoxic activity of these cells. These findings indicated that T-cell subpopulation producing MIF differed from the cytotoxic T-lymphocytes.     

Mechanism of complement-induced stimulation of prostacyclin production by isolated rabbit peritoneum

The interaction between the complement system and prostaglandin synthesis has not thoroughly been explored, although both mediators are known to be involved in inflammatory reactions and endotoxic shock. When rabbit peritoneum, a rich source of prostacyclin forming activity was incubated in serum in which the complement system was activated (CVF, LPS, zymosan), the tissue produced significantly more PGI2, when compared with appropriate controls, indicating that by activation of the complement, factors were generated that stimulated PGI2 biosynthesis. Further results indicated that tryptic cleavage products of complement factor C3 and C5 also led to the appearance of PGI2 releasing principles with a molecular weight of about 7000-11000. The stimulation of PGI2 biosynthesis was explained by enhanced release of AA, and not due to increased activity of cyclo-oxygenase or PGI2 synthetase. Our results suggest that complement-derived products may promote the supply of prostaglandins at the site of inflammation.     

Complement split product C5a mediates the lipopolysaccharide-induced mobilization of CFU-s and haemopoietic progenitor cells, but not the mobilization induced by proteolytic enzymes

Intravenous (i.v.) injection of mice with lipopolysaccharide (LPS), and the proteolytic enzymes trypsin and proteinase, mobilizes pluripotent haemopoietic stem cells (CFU-s) as well as granulocyte-macrophage progenitor cells (GM-CFU) and the early progenitors of the erythroid lineage (E-BFU) from the haemopoietic tissues into the peripheral blood. We investigated the involvement of the complement (C) system in this process. It appeared that the early mobilization induced by LPS and other activators of the alternative complement pathway, such as Listeria monocytogenes (Lm) and zymosan, but not that induced by the proteolytic enzymes, was absent in C5-deficient mice. The mobilization by C activators in these mice could be restored by injection of C5-sufficient serum, suggesting a critical role for C5. The manner in which C5 was involved in the C activation-mediated stem cell mobilization was studied using a serum transfer system. C5-sufficient serum, activated in vitro by incubation with Lm and subsequently liberated from the bacteria, caused mobilization in both C5-sufficient and C5-deficient mice. C5-deficient serum was not able to do so. The resistance of the mobilizing principle to heat treatment (56 degrees C, 30 min) strongly suggests that it is identical with the C5 split product C5a, or an in vivo derivative of C5a. This conclusion was reinforced by the observation that a single injection of purified rat C5a into C5-deficient mice also induced mobilization of CFU-s.     

FURTHER STUDIES ON MOBILIZATION OF CFUs

Mobilization of CFUs from haemopoietic tissues into circulation was studied after injection of different bacterial lipopolysaccharides (LPS), zymosan, phytohaemagglutinin (PHA), concanavalin A (Con A), trypsin and di-isopropyl-fluorophosphate-inhibited trypsin. All bacterial LPS used gave an increase of CFUs in the peripheral blood at 1 h after i.v. injection. Some variation in activity could not be excluded. As with Salmonella typhosa LPS, zymosan gave an increase in circulating CFUs during the first few hr and a second peak a few days later. After injection of zymosan as well as S. typhosa LPS the second peak in the blood was accompanied by a large increase in CFUs numbers in the spleen. PHA gave an immediate mobilization of CFUs, but the mobilization after injection of Con A during the first few hr occurred more slowly. After injection of S. typhosa LPS, zymosan and PHA the blood C3 level was found to be depressed considerably. This might indicate that the complement system is involved in the early mobilization of CFUs. Dexamethasone, a synthetic hormone which has been reported to give sequestration of several cell types in the bone marrow, did not inhibit the early and late mobilization of CFUs which normally occurs after injection of S. typhosa LPS.     

Cell-mediated immunity to mouse adenovirus infection. Macrophage migration inhibition test.

Cell-mediated immunity (CMI) to mouse adenovirus (M-Ad) infection was studied by macrophage migration inhibition test (MMI) as one of in vitro correlates of CMI. Both direct and indirect tests showed clearly that migration of packed peritoneal exudate cells (PEC) (immune mouse or nonimmune guinea pig) was remarkably inhibited; MIF was produced by interactions between immune PEC and infected cell extracts and between immune spleen cells and infected cells or their extracts. The antigen(s) responsible for the above MMI was demonstrated in 6- to 12-hour infected ME cells, and FUdR-treated infected ME cells. Since under these conditions there is S antigen(s) synthesis but not capsid antigen synthesis, the antigen(s) concerned must be an S antigen(s). T cells sensitized to infected cells were shown to be required to induce MMI. The MMI is specific for M-Ad, since no cross MMI was observed between M-Ad and SV40 systems. Time course study of the development of CMI to M-Ad by MMI tests showed that CMI became detectable 4 days post-infection (pi), reached its peak level about 10 days pi, and faded away rapidly in about 10 days thereafter.     

Production of macrophage migration inhibitory factor(s) (MIF) by virus-transformed cells

Rodent cell lines transformed by SV40, polyoma virus and Rous sarcoma virus cultured in vitro release material into the culture medium which inhibits the migration of guinea pig macrophages. Similar macrophage migration inhibitory factors (MIF) were not detected in cell-free supernatants harvested from untransformed cell cultures. Comparison of the MIF produced by virus-transformed cells with MIF derived from peripheral lymphocytes stimulated in vitro by phytohemagglutinin (PHA) revealed that they had similar molecular weights (25 000), heat stability and were both inhibited by α-fucose and lotus agglutinin. Incubation of MIF-containing cell-free supernatants from transformed cells with pancreatic trypsin inhibitor, soybean trypsin inhibitor and diisopropylfluorophosphate eliminated the MIF activity. The possible identity of the MIF released by transformed cells as a protease is discussed with reference to a potential role in modifying the surface properties of transformed cells.     

The Dexamethasone-induced Inhibition of Proliferation, Migration, and Invasion in Glioma Cell Lines Is Antagonized by Macrophage Migration Inhibitory Factor (MIF) and Can Be Enhanced by Specific MIF Inhibitors

Glioblastomas (GBMs) are the most frequent and malignant brain tumors in adults. Glucocorticoids (GCs) are routinely used in the treatment of GBMs for their capacity to reduce the tumor-associated edema. Few in vitro studies have suggested that GCs inhibit the migration and invasion of GBM cells through the induction of MAPK phosphatase 1 (MKP-1). Macrophage migration inhibitory factor (MIF), an endogenous GC antagonist is up-regulated in GBMs. Recently, MIF has been involved in tumor growth and migration/invasion and specific MIF inhibitors have been developed on their capacity to block its enzymatic tautomerase activity site. In this study, we characterized several glioma cell lines for their MIF production. U373 MG cells were selected for their very low endogenous levels of MIF. We showed that dexamethasone inhibits the migration and invasion of U373 MG cells, through a glucocorticoid receptor (GR)- dependent inhibition of the ERK1/2 MAPK pathway. Oppositely, we found that exogenous MIF increases U373 MG migration and invasion through the stimulation of the ERK1/2 MAP kinase pathway and that this activation is CD74 independent. Finally, we used the Hs 683 glioma cells that are resistant to GCs and produce high levels of endogenous MIF, and showed that the specific MIF inhibitor ISO-1 could restore dexamethasone sensitivity in these cells. Collectively, our results indicate an intricate pathway between MIF expression and GC resistance. They suggest that MIF inhibitors could increase the response of GBMs to corticotherapy.     

Activated protein C inhibits tumor necrosis factor and macrophage migration inhibitory factor production in monocytes

The precise regulatory mechanisms of amplification and downregulation of the pro- and anti-inflammatory cytokines in the inflammatory response have not been fully delineated. Although activated protein C (APC) and its precursor protein C (PC) have recently been reported to be promising therapeutic agents in the management of meningococcal sepsis, direct evidence for the anti-inflammatory effect remains scarce. We report that APC inhibits in vitro the release of tumor necrosis factor (TNF) and macrophage migration inhibitory factor (MIF), two known cytokine mediators of bacterial septic shock, from lipopolysaccharide (LPS)-stimulated human monocytes. The THP-1 monocytic cell line, when stimulated with LPS and concomitant APC, exhibited a marked reduction in the release of TNF and MIF protein in a concentration-dependent manner compared to cells stimulated with LPS alone. This effect was observed only when incubations were performed in serum-free media, but not in the presence of 1-10% serum. Serum-mediated inhibition could only be overcome by increasing APC concentrations to far beyond physiological levels, suggesting the presence of endogenous serum-derived APC inhibitors. Inhibition of MIF release by APC was found to be independent of TNF, as stimulation of MIF release by LPS was unaltered in the presence of anti-TNF antibodies. Our data confirm that the suggested anti-inflammatory properties of APC are due to direct inhibition of the release of the pro-inflammatory monokine TNF, and imply that the anti-inflammatory action of APC is also mediated via inhibition of MIF release.     

Discovery of chromenes as inhibitors of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is an essential signaling cytokine with a key role in the immune system. Binding of MIF to its molecular targets such as, among others, the cluster of differentiation 74 (CD74) receptor plays a key role in inflammatory diseases and cancer. Therefore, the identification of MIF binding compounds gained importance in drug discovery. In this study, we aimed to discover novel MIF binding compounds by screening of a focused compound collection for inhibition of its tautomerase enzyme activity. Inspired by the known chromen-4-one inhibitor Orita-13, a focused collection of compounds with a chromene scaffold was screened for MIF binding. The library was synthesized using versatile cyanoacetamide chemistry to provide diversely substituted chromenes. The screening provided inhibitors with IC₅₀’s in the low micromolar range. Kinetic evaluation suggested that the inhibitors were reversible and did not bind in the binding pocket of the substrate. Thus, we discovered novel inhibitors of the MIF tautomerase activity, which may ultimately support the development of novel therapeutic agents against diseases in which MIF is involved.     

Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC₅₀ values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro¹; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.     

Evidence for a surface receptor for human migration inhibitory factor on a B-lymphoid cell line.

Reception by PGLC-33H target cells for the migration inhibitory factor (MIF) produced by this established line has been investigated by pulse time and temperature dependence, MIF absorption, and abrogation by trypsinization. PGLC-33H supernatants containing MIF were concentrated 5× with Carbowax and dialyzed against serum free RPMI-1640 before use. Prior to standard capillary migration assay a minimum 30 min pulse of MIF at 37 °C is required for significant migration inhibition (MI > 20%). No significant MI is observed when cells are pulsed at 4 °C for up to 2 hr. Preincubation with PGLC-33H for 1 hr at 37 °C reduces activity of supernatants from 38 to 13% MI; at 4 °C to 27% MI. Trypsinization of target cells for 30 min at 25 °C abrogates response to MIF (43 to ?14% MI). Trypsinized cells did not reduce activity of supernatants. MIF activity is abolished (32 to 3% MI) in samples preincubated with supernatants of the trypsinized cells inactivated with serum. These data suggest that cells from the human B-lymphoid cell line PGLC-33H have a surface receptor for human MIF.