Fractionation of murine macrophage inhibitory factor into two distinct species

Murine migration inhibitory factor (MIF) produced by concanavalin A-stimulated lymph node cells from C57BL/6 mice was fractionated by Sephadex G-100 gel filtration, density gradient electrophoresis, and isoelectrofocusing in a sucrose density gradient and assayed on in vitro-cultivated bone marrow macrophages from C57BL/6 mice. Two major MIF species, pH3-MIF with an isoelectric point of 3.0–4.3 and pH5-MIF with an isoelectric point of 4.6 to 5.2, were obtained. The similarity of murine MIF to guinea pig and human MIF is discussed.     

The effect of tunicamycin on the molecular heterogeneity of guinea pig migration inhibitory factor

Migration inhibitory factor (MIF) was produced by guinea pig lymph node cells stimulated with concanavalin A in the absence and presence of the glycosylation inhibitor tunicamycin. The active supernatants were purified on Sephadex G-100 and fractionated into pH 3-MIF and pH 5-MIF using isoelectrofocusing. When produced in the absence of tunicamycin pH 3-MIF shows extensive charge heterogeneity with activity focusing from pH 3.0 to 4.5; it elutes from Sephadex G-75 with molecules of an apparent MW of 70,000. In contrast, pH 3-MIF produced in the presence of tunicamycin (TM-pH 3-MIF) focuses as a sharp homogeneous peak with a pI of 3.6 to 4.0 and elutes from Sephadex G-75 with molecules of an apparent MW of 25,000–35,000. TM-pH 3-MIF is trypsin sensitive and displays a buoyant density similar to that of proteins which contain little or no carbohydrate (?₂₅ 1.26?1.34). Tunicamycin caused no detectable change in the characteristics of pH 5-MIF. This study indicates that lymphocytes stimulated in the presence of tunicamycin produce a novel species of pH 3-MIF with characteristics distinct from classical pH 3-MIF.     

Generation of human hybridomas producing migration inhibitory factor (MIF) and of murine hybridomas secreting monoclonal antibodies to human MIF

Human T-cell hybridomas were established by hybridization of concanavalin A (Con A)-stimulated human peripheral blood T lymphocytes with cells from a 6-thioguanine-resistant, aminopterin-sensitive mutant line designated CEM-WH4, derived from the continuously growing human T cell line, CEM. High levels of MIF activity were demonstrated in the supernatants of two hybridoma lines, T-CEMA and T-CEMB but not of CEM-WH4 when stimulated with phorbol myristate acetate and phytohemagglutinin. In comparison, MIF derived from Con A-stimulated peripheral blood mononuclear cells showed 100 times less activity. Upon isoelectrofocusing, MIF activity of T-CEMB was found exclusively between pH 4.6 and 5.3 whereas MIF derived from T-CEMA showed heterogeneity with a major peak of MIF recovered at pH 4.6-5.3 and a minor peak at pH 2.4-3.3. These molecules, however, were all found to have an apparent MW of 68,000 and were resistant to trypsin. Most of these characteristics are in accordance with second day pH 3- and pH 5-MIF derived from peripheral blood mononuclear cells. When spleen cells from BALB/c mice immunized with T-CEMB-MIF were used to fuse with NS-1 mouse myeloma cells, nine hybridomas secreting antibodies to human MIF were obtained. Clone D112 which demonstrated the highest MIF-neutralizing activity was found to neutralize MIF derived from T-CEMA, peripheral blood mononuclear cells, and a T cell line, Mo.     

Two migration inhibitory factors with different chromatographic behavior and isoelectric points.

Migration inhibitory factor (MIF), produced by stimulation of guinea pig lymph node cells with concanavalin A, was fractionated by Sephadex G-100 gel filtration, sucrose density gradient electrophoresis, and isoelectrofocussing. Two distinct species were identified and separated. One, pH 3-MIF, has an isoelectric point of 3.0 to 4.5 and elutes from Sephadex G-75 columns with molecules having an apparent m.w. of 65,000 (Kd of 0.05 to 0.12). The other, pH 5-MIF, has an isoelectric point of 5.0 to 5.5 and elutes with molecules having an apparent m.w. between 25,000 and 43,000 (Kd of 0.15 to 0.23).     

Production of migration-inhibitory factor by a human T-lymphoblast cell line

Migration-inhibitory-factor (MIF) activity was detected in culture supernatants of the human T-lymphoblast cell line Mo after stimulation with phytohemagglutinin and phorbol myristate acetate. MIF activity was not detected in unstimulated cultures reconstituted with phytohemagglutinin and phorbol myristate acetate. Conditioned medium from the cell line Mo was fractionated by Sephadex G-100 gel nitration. MIF-containing Sephadex fractions corresponding to a M_r, of 60,000 to 70,000 were further fractionated by isoelectrofocusing, resulting in a sharp peak of activity with a pI of 4.6 to 5.2. This MIF species constitutes a major form secreted by Mo cells; it adheres to Con A-Sepharose, is trypsin-resistant, and is denser than pure protein as determined by CsCl density gradient centrifugation. These are the same physicochemical characteristics previously established for second-day pH5-MIF from peripheral blood mononuclear cells (W. Y. Weiser et al., J. Immunol.126, 1958, 1981). In contrast, Sephadex fractions corresponding to larger molecules (M_r 70,000–90,000) contain at least two additional MIF species. These larger MIF forms have a pI of 3.0 to 3.5 and of 4.6 to 5.2 and lack affinity to Con A-Sepharose. Thus, the Mo T-cell line produces large quantities of at least three different species of human MIF.     

Partial purification and characterization of a human lymphokine which induces antileishmanial activity in mouse macrophages

Human blood mononuclear cells stimulated in vitro with concanavalin A secrete a lymphokine which activates mouse macrophages to kill Leishmania enriettii. Supernatants of cells cultivated for 1 day and for 2 days were analyzed for this activity after G-100 gel filtration and isoelectrofocusing. First day antileishmanial activity was found in a MW range of 23,000 with an isoelectric point of 3.6 to 4.0. Second day antileishmanial activity eluted in a peak in the MW range of 23,000 to 65,000 with an isoelectric point of 4.0 to 4.3. This activity could be completely separated from migration-inhibitory factor (MIF) after isoelectrofocusing in first day and in second day supernatants. Antileishmanial activity could be separated from interferon-gamma in first day, but not in second day supernatants.     

Human T-lymphocyte chemotactic activity: nature and production in response to antigen

Previous studies have shown that supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells and isolated Leu-2 suppressor/cytotoxic T cells are chemotactic for Leu-3 helper/inducer T cells. The current study shows that lymphocyte chemotactic factor (LCF) is also produced following antigen (tetanus toxoid) challenge of mononuclear cells obtained from recently immunized human donors. LCF was detected in 24-hr supernatants from mononuclear cells challenged with tetanus and was produced maximally at 48 hr. Tetanus toxoid challenge of mononuclear cells obtained from individuals whom had not received a tetanus immunization for 7 to 10 years prior to testing showed little or no production of LCF. Serial studies of these individuals following a tetanus booster immunization showed that LCF was produced by antigen-challenged mononuclear cells obtained 1-5 days postimmunization, was produced optimally 5-15 days postimmunization, and was still produced by antigen-challenged mononuclear cells obtained 6 weeks later. Fractionation of mononuclear cells from immunized donors into glass wool nonadherent lymphocytes, T lymphocytes, and non-T lymphocytes showed that tetanus-toxoid-induced LCF was produced by nonadherent lymphocytes and T cells but not non-T cells. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T-cell subpopulations showed that LCF production by antigen-challenged isolated subpopulations was limited to the Leu-2 suppressor/cytotoxic T-cell subset. Characterization of both Con A and tetanus toxoid-induced LCF by gel filtration on Sephadex G-100 demonstrated the presence of two peaks of LCF corresponding to molecular weights of approximately 14,000-17,000 and 40,000-50,000.     

A phorbol ester (TPA) can replace macrophages in human lymphocyte cultures stimulated with a mitogen but not with an antigen

A culture system was developed in which human peripheral blood mononuclear cells (PBM) depleted of macrophages did not proliferate in response to the lectin mitogen PHA or to the soluble antigen of tetanus toxoid. These cells were able to respond to both mitogen and antigen if purified autologous macrophages were added back to the culture. The response to PHA was partially restored by supplementing the cultures with supernatants from LPS-stimulated macrophages or with partially purified human interleukin 1 (IL 1). The response to tetanus was not restored by reconstitution with these materials. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), has been shown to have IL 1-like effects in other species and is a polyclonal activator of human T and B lymphocytes. In this study, we tested the ability of TPA to replace macrophages in human lymphocyte cultures stimulated with mitogen or with antigen. Small doses of TPA (50 ng/ml) completely replaced macrophages in the PHA-stimulated cultures; however, in doses of up to 400 ng/ml, TPA was not able to replace macrophages in cultures stimulated with tetanus. Thus, TPA appears to mimic the macrophage-replacing ability of soluble factors (IL 1, macrophage supernatants) in the triggering of human lymphocytes.     

Dissociation of human interferon-gamma-like activity from migration-inhibition factor

Supernatants harvested from concanavalin A-stimulated human peripheral mononuclear cells after 24 hr of incubation contain one interferon species similar to human interferon-gamma (IFN-γ) with a pI of 4.6–5.3 (first day pH 5 IFN-γ). In contrast, during the subsequent 24 hr of incubation two species with properties of IFN-γ are produced with pI of 3.6–4.0 (second day pH 4 IFN-γ) and 4.6–5.6 (second day pH 5 IFN-γ), respectively. First day pH 5 IFN-γ and second day pH 5 IFN-γ have been found to differ on the basis of trypsin sensitivity. This pattern of polymorphism is similar to the pattern previously described for human migration-inhibitory factor (MIF) which can be separated into first day pH 5 MIF, second day pH 3 MIF, and second day pH 5 MIF. However, IFN-γ-like species can be differentiated from MIF biochemically and antigenically. Fractions with second day pH 4 IFN-γ have no MIF activity and fractions with second day pH 3 MIF contain no IFN activity. In addition, first and second day pH 5 MIF, which also contain IFN-γ activity, can be separated from the latter by precipitation as well as neutralization with polyclonal and monoclonal anti-human MIF antibodies.     

Modified Radioimmunoassay for Murine Sarcoma-Leukemia Virus Group-Specific Antigen

Iodination of disrupted Moloney strain murine sarcoma-leukemia virus resulted in labeled group-specific (gs) protein which was subsequently purified on an isoelectrofocusing column. This iodinated purified gs antigen, prepared from a relatively small quantity of purified virus, was used in a radioimmunoassay. A radioimmunoassay inhibition method was developed so that antibody specific for mammalian C-type gs antigen could be measured in undiluted or low dilutions of test serum without altering the known reagents of the test. The gs antigen isolated from purified Moloney strain murine sarcoma-leukemia virus has an isoelectric point (pH 5.95) which is significantly lower than that reported for other murine leukemia viruses.     

In vitro human antibody production to the Haemophilus influenzae type b capsular polysaccharide

In vitro production of human antibody to the Haemophilus influenzae type b capsular polysaccharide (PRP) and to tetanus toxoid (TT) and diphtheria toxoid was measured in culture supernatants of peripheral blood mononuclear cells and by enumeration of antibody secreting cells (AbSC) in an enzyme-linked immunosorbent-plaquing assay. Normal adult peripheral blood mononuclear cells stimulated with Epstein-Barr virus secreted anti-PRP antibody with a frequency of 1/552 to 1/1190 relative to total Ig secreting cells; the frequency of AbSC to tetanus toxoid (TT) was 7.5 times higher (p less than 0.05). These frequencies did not change significantly after in vivo immunization, although the isotype distribution shifted toward increased IgG for TT and increased IgG and IgA for PRP. At 8 days postimmunization, spontaneous AbSC to PRP and TT were detected; frequencies for total anti-TT AbSC again being higher than anti-PRP, but there were significantly more IgA plaques among anti-PRP AbSC. Spontaneous AbSC were suppressed in culture by pokeweed mitogen and enhanced by cyclosporine. Three wk after in vivo immunization with PRP and TT, in vitro stimulation with pokeweed mitogen, Staphylococcus aureus Cowan 1 bacteria, or antigen induced anti-TT but not anti-PRP in vitro antibody secretion, although Epstein-Barr virus induced both. These data suggest that PRP, a polysaccharide, and TT, a protein, differ in their requirements for in vitro activation with antigen and mitogens.     

Human T4+ lymphocytes produce a phagocytosis-inducing factor (PIF) distinct from interferon-alpha and interferon-gamma

Unstimulated peripheral blood mononuclear cells from patients with angiocentric T cell immunoproliferative disorders and concanavalin A-stimulated normal peripheral blood mononuclear cells secrete a phagocytosis-inducing factor (PIF) that induces a fivefold to 50-fold enhancement of phagocytosis of IgG-coated ox red blood cells by U937 cells. We investigated the identity, production, and mechanism of the action of PIF. PIF activity was demonstrated in supernatants from nine of 44 phytohemagglutinin-stimulated interleukin 2 (IL 2)-dependent T cell lines and clones derived from purified T4+ cells, but was not found in supernatants from 26 lines and clones derived from phytohemagglutinin-stimulated T8+ cells. In addition, PIF was produced by four of four antigen-specific T cell lines and clones after stimulation with the appropriate antigen and antigen-presenting cells, and by HUT-102, a human T cell lymphotropic virus type I-transformed T cell line. PIF from all of these sources caused significant inhibition of U937 proliferation. This proliferation-inhibiting activity co-purified with phagocytosis-enhancing activity in sizing procedures and isoelectric focusing, which yielded an estimated m.w. of 35,000 to 55,000 and an estimated isoelectric point of 5.0 to 6.0 for PIF. In contrast, IL 2, recombinant interferon-alpha, and recombinant interferon-gamma had no effect on phagocytosis by U937 cells, and antibodies to interferon-alpha and interferon-gamma did not block the phagocytosis-inducing activity of PIF-containing supernatants. PIF appears to be a distinct lymphokine produced by a subset of T4+ lymphocytes, possibly those that proliferate in response to antigen. PIF may be important in the induction of erythrophagocytosis, which is associated with certain T cell immunoproliferative disorders.     

In vitro immunization of human lymphocytes. I. Production of human monoclonal antibodies against bombesin and tetanus toxoid

A procedure for in vitro sensitization of human lymphocytes against bombesin conjugated to tetanus toxoid (BTT) is described. Bombesin is a tetradecapeptide associated with small cell lung carcinoma. We found that antibody responses against bombesin as well as tetanus toxoid could be generated in vitro by culturing nylon-separated human splenic lymphocytes for 6 days with lipopolysaccharide, phytohemagglutinin-activated lymphocyte supernatants, human AB serum, and bombesin conjugated to tetanus toxoid. Cells sensitized by this procedure were fused to murine myeloma cells, NS-1. The specificities of resulting hybrids were analyzed by enzyme-linked immunoassays and competitive inhibition experiments. Hybrids secreting anti-bombesin (IgM) or anti-tetanus toxoid (IgM or IgG) were obtained. The ratio of IgG to IgM antibodies against tetanus toxoid could be increased by using antigen coupled to Sepharose beads. The sensitization procedure described here offers a system for the study of antigenic stimulation of human B lymphocytes in vitro and for the production of human monoclonal antibodies with the desired specificities.     

Glutathione S-transferase, similar to sigma class, from skin secretions of Xenopus laevis

Using glutathione affinity chromatography followed by isoelectrofocusing, we purified from the skin secretion of Xenopus laevis an isoenzyme of glutathione S-transferase with an apparent subunit molecular mass of 22.5 kDa and an isoelectric point at pH 5.1. Its N-terminal amino acid sequence was highly similar to that of the sigma class glutathione S-transferase, which previously was demonstrated to have a glutathione-dependent prostaglandin D2 synthase activity. Immunohistochemistry analysis revealed that the isoenzyme was located in the cytoplasm of granular gland cells.     

Cyclophosphamide-Induced Cystitis Increases Bladder CXCR4 Expression and CXCR4-Macrophage Migration Inhibitory Factor Association

Background

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in cystitis and a non-cognate ligand of the chemokine receptor CXCR4 in vitro. We studied whether CXCR4-MIF associations occur in rat bladder and the effect of experimental cystitis.

Methods and Findings

Twenty male rats received saline or cyclophosphamide (40 mg/kg; i.p.; every 3^rd day) to induce persistent cystitis. After eight days, urine was collected and bladders excised under anesthesia. Bladder CXCR4 and CXCR4-MIF co-localization were examined with immunhistochemistry. ELISA determined MIF and stromal derived factor-1 (SDF-1; cognate ligand for CXCR4) levels. Bladder CXCR4 expression (real-time RTC-PCR) and protein levels (Western blotting) were examined. Co-immunoprecipitations studied MIF-CXCR4 associations.Urothelial basal and intermediate (but not superficial) cells in saline-treated rats contained CXCR4, co-localized with MIF. Cyclophosphamide treatment caused: 1) significant redistribution of CXCR4 immunostaining to all urothelial layers (especially apical surface of superficial cells) and increased bladder CXCR4 expression; 2) increased urine MIF with decreased bladder MIF; 3) increased bladder SDF-1; 4) increased CXCR4-MIF associations.

Conclusions

These data demonstrate CXCR4-MIF associations occur in vivo in rat bladder and increase in experimental cystitis. Thus, CXCR4 represents an alternative pathway for MIF-mediated signal transduction during bladder inflammation. In the bladder, MIF may compete with SDF-1 (cognate ligand) to activate signal transduction mediated by CXCR4.     

Antigen presentation by Hodgkin's disease cells

The L428 tumor cell line is a long-term tissue culture of Reed-Sternberg cells which was derived from the pleural effusion of a patient with Hodgkin's disease. The L428 cells express all known cell surface antigens, cytochemical staining, and cytologic features of freshly explanted Reed-Sternberg cells. In addition to the previously described HLA-DR cell surface antigens, the L428 cells are now demonstrated to express both DS and SB alloantigens. Thus, the L428 cells express all of the known subclasses of the human immune response genes that are located in the major histocompatibility complex. Furthermore, the L428 cells are capable of presenting soluble antigen to T cells in a genetically restricted fashion. T cell lines were established from normal donors previously immunized with tetanus toxoid. The T cells utilized were incapable of tetanus toxoid-induced proliferation unless antigen-presenting cells were added to the cultures. However, T cells from the two normal donors, which like the L428 cells expressed HLA-DR 5, demonstrated significant proliferative responses when cultured with tetanus toxoid and L428 cells. No proliferative response was observed when the L428 cells were used as antigen-presenting cells for a DR (4,-), DR (2,-) or DR (1,7) T cell line. The tetanus toxoid dose-response curve was similar regardless of whether autologous mononuclear leukocytes or L428 cells were used as antigen-presenting cells. The T cell proliferation induced by soluble antigen was also blocked by anti-HLA-DR antibody. Thus, functionally, Hodgkin's disease may be classified as a tumor of antigen-presenting cells.     

Purification of Cytochrome b5 from Pig Testis Microsomes by Isoelectric Focusing in an Immobiline pH Gradient

Testicular cytochrome b5 was purified by a procedure including preparative isoelectrofocusing. The cytochrome b5 was determined to have an isoelectric point of 4.45 on analytical isoelectric focusing. The purified cytochrome b5 was found to be homogeneous and its molecular weight was estimated to be 16,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The oxidized and reduced forms of the purified preparation exhibited absorption spectra of a typical cytochrome b5. A 69-fold purification was achieved with an overall yield of 6.2%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing column chromatography and preparative isoelectric focusing.     

The cellular basis for differential lymphokine responses to mitogen stimulation

Human mononuclear cells from some individuals produce macrophage migration inhibition factor (MIF) when stimulated with Con A while those of others produce migration stimulation factor (MStF). T cells were responsible for these different responses but T4 cells produced MIF and T8 cells produced MStF regardless of the global response which was not explained by the individual T4:T8 ratios. Admixing the T-cell subpopulations in vitro revealed that MIF responses switched to MStF responses between T4:T8 ratios of 75:25 and 50:50 with MStF responders switching at higher ratios than MIF responders. Pulse exposure to supernatants from Con A-stimulated T4-enriched cells significantly reduced migration indices resulting from stimulation of fresh cells, promoting MIF responses regardless of the responder status of the supernatant donor. In contrast, supernatants from T8-enriched cells, when obtained from MStF responders, significantly increased migration indices while there was no effect when the supernatants were obtained from MIF responders. These results suggest that soluble factors from T8 cells are primarily responsible for determining whether an individual mounts a MIF or MStF response to Con A stimulation.     

The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major

The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune to cutaneous leishmaniasis. Peripheral blood mononuclear cells from Sudanese individuals with a past history of self-healing cutaneous leishmaniasis proliferated vigorously in response to PSA-2 isolated from Leishmania major, whereas the antigen did not activate cells from presumably unexposed Danes. Peripheral blood mononuclear cells from individuals with previous L. major infection had varying proliferative responses to PSA-2 derived from L. donovani promastigotes. Peripheral blood mononuclear cells activated by PSA-2 from L. major produced high amounts of interferon-γ and tumour necrosis factor-β, and little interleukin-4, thereby showing a Th1 cytokine pattern. Parallel cultures showed clear Th1 and Th2 response patterns to purified protein derivative of tuberculin or tetanus toxoid, respectively. Flow cytometric analysis revealed that PSA-2 induced blastogenesis in the CD3 positive population and that these cells were the major source of interferon-γ. The results show that Th1-like cells recognising PSA-2 are expanded during infection by L. major and that they maintain their Th1-like cytokine profile upon reactivation in vitro. Since immunity to cutaneous leishmaniasis is mediated by antigen-specific Th1-like cells, PSA-2 might be considered a vaccine candidate for human leishmaniasis.     

Serum antigens of cattle. VI. Isolation and properties of an intracellular antigen (Eel) from cattle erythrocytes*

The erythrocyte antigen (Ecl) of cattle has been isolated using DEAE-cellulose chromatography, Sephadex gel filtration, and isoelectrofocusing. The purified antigen is free from hemoglobin and migrates as a single protein on polyacrylamide gels. It exhibits an absorption maximum at 278 nm and has a molecular weight of about 100 000; its isoelectric point is 4.95. The antigen has a carbohydrate moeity and loses its activity after oxidation with periodate. The evidence suggests that it is not a common isozyme, but its exact identity remains unknown.