Biomedical subjects

C L Geczy

Publications and source records attributed to C L Geczy.

At least 55 records · Page 3Linked to original sources

PubMed1991

Discordant expression of tissue factor antigen and procoagulant activity on human monocytes activated with LPS and low dose cycloheximide.

The mechanisms underlying the superinduction of procoagulant activity by cycloheximide (CHX) on LPS-activated human monocytes have been investigated. Tissue factor (TF) activity of intact, viable cells was quantitated with a plasma recalcification assay and assays using chromogenic substrates specific for thrombin and factor Xa (FXa). TF antigen was measured simultaneously by immunocytochemical staining and immunoblotting with an anti-TF monoclonal antibody (MAb). Peripheral blood mononuclear cells (PBMC) activated with LPS in the presence of low dose CHX expressed more TF activity (approx. 100% increase) than cells activated with LPS alone. However, TF antigen levels were decreased approximately 70% by CHX. This discordant relationship was due primarily to differences in rates of activation of factor X (FX); LPS/CHX-treated PBMC activated nearly twice as much FX as LPS-treated cells (2.19 +/- 0.37 versus 1.10 +/- 0.21 ng FXa/10(6) PBMC/min, respectively). These studies indicate that TF cofactor activity on LPS/CHX-treated monocytes was approximately 7 times greater than that present on LPS-treated cells. Increased TF functional activity may be due to CHX-induced alterations in the type and content of phospholipids (PL) in the cell membrane. Results showed that exogenous mixed PL markedly increased TF activity on LPS-activated monocytes, but not on LPS/CHX-activated cells, without increasing TF antigen levels or altering cell viability. Membrane alterations may occur on monocytes in certain pathological or iatrogenic conditions resulting in a highly active form of TF in vivo.

Antibodies, Monoclonal↗

PubMed1991

Radioimmunoassay for the detection of active-site specific thrombin inhibitors in biological fluids. I. Assay characteristics and quantitation of recombinant hirudin.

A sensitive radioimmunoassay (RIA) for the quantitation of recombinant (r) hirudin in biological fluids is described. Taking advantage of the highly specific hirudin-thrombin interaction, a monoclonal antibody to human alpha-thrombin was used to capture hirudin-thrombin complexes in a competitive binding assay. Quantitation of r.hirudin in buffer, plasma or urine at concentrations ranging from 0.17 to 20 ng/ml (1.7 x 10(-3) to 2 x 10(-2) antithrombin units/ml) was achieved. In the absence of competing unlabelled r.hirudin the assay also measured alpha-thrombin (from 2 x 10(-4) to 1 x 10(-2) NIH units/ml) in citrated or defibrinated human plasma. A series of peptides corresponding to the carboxyl-terminal region of hirudin and with varying anticoagulant activities did not displace 125I-r.hirudin in the RIA described, confirming published data that these hirudin fragments bind to a site distant to the catalytic site of thrombin. The assay was used to test hirudin clearance after bolus i.v. injections of 0.1 mg r.hirudin [Val1-Val2] into human volunteers. The plasma concentrations and elimination kinetics of r.hirudin were in good agreement with published data and a close correlation between hirudin plasma concentration and prolonged clotting time was observed.

Antibodies, Monoclonal↗

PubMed1991

Radioimmunoassay for the detection of active-site specific thrombin inhibitors in biological fluids. II. Heparin affects the binding of hirudin to alpha-thrombin.

A radioimmunoassay for recombinant (r.) hirudin was used to study the effect of heparin and other glycosaminoglycans (GAG's) on interactions between hirudin and thrombin. Binding of 125I-r.hirudin to thrombin in the presence or absence of heparin (and other GAG's) was monitored by immunoprecipitation of thrombin-125I-r.hirudin complexes with a monoclonal antibody to human a-thrombin. Heparin and dextran sulfate substantially reduced hirudin binding to thrombin in human plasma. Inhibition by heparin was restored by addition of increasing amounts of antithrombin (AT) to samples containing constant amounts of heparin, thrombin and 125I-r.hirudin and was not neutralized by protamine sulfate. At very low heparin concentrations competitive displacement of 125I-r.hirudin was observed, while in the presence of heparin-AT complexes some 125I-r.hirudin remained bound to thrombin, suggesting that two or more binding sites for hirudin on the thrombin molecule may be occupied simultaneously.

Binding Sites↗

PubMed1991

Characterisation of monoclonal antibodies to human factor X/Xa. Initial observations with a quantitative ELISA procedure.

Monoclonal antibodies to human plasma factor X (FX) and factor Xa (FXa) have been developed using several modifications of previously described techniques. These include the use of footpad immunisation with a suspension of free and nitrocellulose-bound antigen with subsequent fusion of popliteal lymph node cells. From a panel of 17 reactive hybridomas to FX, 3 were selected for further characterisation. An additional hybridoma reactive to FXa but not FX was also selected. Two monoclonal antibodies designated FX52 and FX64 were specific for FX with no reactivity to FXa, while antibody FXa24 was specific for FXa. Another FX/FXa95 reacted with both FX and FXa. All selected antibodies were of the IgG isotype and reacted both with native antigen and antigen transferred to nitrocellulose by Western blotting. Initial observations suggest that Mab FX52 may be used to quantitate FX levels in plasma.

Animals↗

PubMed1991

Arachidonic acid metabolites in normal and autoimmune mice do not influence lymphocyte-high endothelial venule interactions.

In peripheral lymphoid organs the number of lymphocytes and the proportion of functional lymphocyte subsets are regulated by multiple factors including the control of lymphocyte migration by selective lymphocyte-high endothelial venule (HEV) interactions. In this study, prostaglandin E2 (PGE2) levels from normal and autoimmune mouse lymph node cells were measured. The contribution of eicosanoids to lymphocyte-HEV interactions in normal (CBA/T6) and autoimmune (MRL/n) mice was examined. There was no association between PGE2 production in normal or autoimmune mice and the age of onset of disease activity in the latter strains. Arachidonic acid metabolites, in particular PGE2 and leukotriene B4 (LTB4), did not have any effects on lymphocyte-HEV binding. Likewise, lymphocytes treated in vivo and/or in vitro with arachidonic acid metabolite inhibitors (acetyl salicylic acid, indomethacin, BW755C) did not alter lymphocyte-HEV binding interactions in both normal and autoimmune mice. No clinical significance could be attributed to lymph node PGE2 production and the age of onset of autoimmune disease. In summary, these findings cast doubt on the role of arachidonic acid metabolites in lymphocyte-HEV binding interactions.

Animals↗

PubMed1991

Actinomycin D upregulates lipopolysaccharide induction of macrophage procoagulant expression and tumour necrosis factor-alpha production.

The antitumour antibiotic actinomycin D (Act D) and the aminosugar D-galactosamine both enhance the sensitivity of animals to bacterial lipopolysaccharide (LPS). Lipopolysaccharide stimulates macrophage membrane-bound procoagulant activity (MPCA) and tumour necrosis factor-alpha (TNF-alpha) production in vitro. We investigated the effects of LPS combined with either Act D or D-galactosamine on procoagulant and TNF-alpha production in vitro. Actinomycin D directly induced procoagulant on the malignant monocytoid cell line WEHI 265, and synergized with LPS to enhance MPCA on both WEHI 265 cells and thioglycollate-induced peritoneal exudate macrophages. In the presence of Act D, exudate macrophages expressed procoagulant in response to concentrations of LPS 100,000-fold lower than normally required. Pulsing experiments demonstrated that LPS primed these cells within 4 h to respond to Act D, whereas 4 h priming with Act D inhibited subsequent procoagulant induction by LPS. Although its effects on TNF-alpha production were less intense, low levels of Act D more than doubled TNF-alpha produced by LPS-stimulated exudate macrophages. Procoagulant expression and TNF-alpha production were not always co-ordinately expressed; interferon-gamma (IFN-gamma) synergized with LPS to enhance both responses but when IFN-gamma was combined with Act D only procoagulant was upregulated. D-galactosamine failed to affect these macrophage responses. Results indicate different in vivo mechanisms of enhancement of LPS toxicity by these two agents.

Animals↗

PubMed1990

Induction of macrophage procoagulant expression by cisplatin, daunorubicin and doxorubicin.

Cisplatin, doxorubicin and daunorubicin (drugs which intercalate with DNA) influenced the membrane-bound procoagulant potential of murine thioglycollate-induced peritoneal exudate (TG-PEC) macrophages and the monocytoid cell line WEHI 265, whereas the antimetabolites 5-fluorouracil and methotrexate had no effect. Enhanced procoagulant was not caused by non-specific toxicity of these agents. Cisplatin directly increased the procoagulant expressed on WEHI 265 cells, whereas MPCA on TG-PEC was enhanced only when cisplatin was combined with a second stimulant, either bacterial lipopolysaccharide (LPS) or interferon (IFN gamma). WEHI 265 cells failed to respond to the anthracycline drugs, either alone or in combination with LPS, whereas they enhanced the IFN gamma response. Doxorubicin and daunorubicin increased the LPS response of TG-PEC by approximately 4-fold and the IFN gamma response by approximately 10-fold. Pulsing experiments suggested that the anthracyclines enhanced procoagulant expression by a mechanism different from cisplatin. Daunorubicin primed TG-PEC within 4 hr to respond to low levels of LPS, whereas either LPS or cisplatin primed these cells to respond to cisplatin or LPS respectively. Furthermore, the procoagulant expressed by TG-PEC stimulated by LPS/cisplatin had properties of tissue factor (TF: 50% total activity) and Factor VIIa (50% total procoagulant)-like activities, whereas the predominant procoagulant on LPS/anthracycline activated TG-PEC was TF-like (70% total activity) with weak Factor VIIa and prothrombinase-like properties.

Animals↗

PubMed1990

Macrophage procoagulant activity as an assay of cellular hypersensitivity to gluten peptides in coeliac disease.

Gluten and casein digests were tested for their ability to induce a cellular immune response on peripheral blood mononuclear cells (PBM) obtained from coeliac patients as well as healthy volunteers and disease control patients using the macrophage procoagulant assay. PBM from coeliac patients who had been on a gluten-free diet for less than 2 years responded strongly to gluten peptides, while coeliac patients at diagnosis or who had maintained a strict gluten-free diet for longer than 5 years showed weaker responses. PBM from healthy volunteers did not respond to gluten or casein peptides, whereas those from patients with Crohn's disease displayed weak reactivity to gluten and casein peptides. Our study using the macrophage procoagulant assay confirms previous findings that lymphoid cells from patients with coeliac disease exhibit a specific cellular immune response to gluten. This assay represents an alternative measure for cell-mediated immunity and is technically much simpler than the previously described leucocyte migration inhibition assay. Macrophage procoagulant activity is measured using a simple plasma recalcification time assay or spectrophotometrically using commercially available chromogenic substrates.

Adult↗

PubMed1990

Effect of locally administered heparins on delayed-type hypersensitivity reactions.

Inflammatory reactions involved in delayed-type hypersensitivity (DTH) are associated with extravascular coagulation and fibrin deposition. Heparin and other anticoagulants administered systemically inhibit DTH reactions but the direct effect of intradermally injected heparin on the development of DTH skin responses has not been reported. The effects of heparin on the DTH reaction elicited by ovalbumin (OVA) in guinea pigs 1-3 weeks after sensitization were examined. Unfractionated heparin, low affinity heparin (LAH; non-anticoagulant) and high affinity heparin (HAH; anticoagulant) were injected together with suboptimal amounts of OVA. Heparin and LAH enhanced skin induration, LAH (0.5 micrograms) by an average of 50% above that due to OVA alone at 24 h (p less than 0.01). In contrast, HAH (0.5 micrograms) significantly reduced skin induration at 24 h. Heparin and LAH also significantly increased cellular infiltration with LAH having the greater effect. At 4 h the infiltrate consisted mainly of neutrophils whereas at 24 h mononuclear cells predominated. Fibrin deposition, assessed both by immunohistology and quantitation of radioactive fibrin extracted from skin test sites, was increased by 30% when OVA was tested in the presence of LAH. Mast cell heparin released locally at sites of DTH has the potential to modulate these reactions in either a pro- or anti-inflammatory manner. This study is the first to demonstrate differences in the capacities of LAH and HAH to modulate cell-mediated inflammation.

Animals↗

PubMed1990

Aberrant lymphocyte migration patterns in systemic lupus erythematosus (MRL/l, MRL/n) mice are independent of the micro-environment.

Mice with systemic lupus erythematosus (SLE) have unusual patterns of lymphocyte traffic characterised by diminished uptake of intravenously injected autoimmune cells into lymph nodes. This study examines the influence of the lymphocyte micro-environment on this aberrant migratory behaviour. To evaluate lymph node lymphocyte-endothelial interactions which can affect lymphocyte distribution without the in vivo influence of liver and spleen, the in vitro high endothelial venule (HEV) binding assay was used. Lymph node HEV binding of autoimmune MRL-lpr/lpr (MRL/l) and MRL(-)+/+ (MRL/n) lymphocytes was increased when compared with CBA/T6 lymphocytes and contrasted with diminished lymph node uptake noted in vivo. This was independent of the lymph node source (MRL/l, MRL/n, CBA/T6) onto which the lymphocytes were overlaid. To examine the influence of the microenvironment on in vivo traffic, 21Cr-labelled lymph node cells from normal CBA/T6 mice were injected into sex-matched MRL/l, MRL/n and CBA/T6 recipients. The distribution of cells was the same in each recipient strain suggesting that the micro-environment had little influence on the lymphocyte trafficking profiles of autoimmune mice. This study supports the view that aberrant lymphocyte migration in autoimmune mice results from defects intrinsic to the lymphocyte population and not the micro-environment.

Age Factors↗

PubMed1990

Thrombin and factor Xa enhance the production of interleukin-1.

Interleukin-1 (IL-1) plays a major role in inflammatory responses. Activation of coagulation and fibrin deposition typical of these reactions is mediated by macrophage procoagulants induced on stimulated macrophages. IL-1 activity in the supernatant of lipopolysaccharide (LPS)-stimulated guinea-pig macrophages was markedly enhanced by the presence of thrombin during macrophage activation. Although thrombin alone had no effect, inclusion of 1 mU/ml of thrombin with suboptimal levels of LPS produced a 200-fold increase in IL-1 activity, and further enhancement was observed with increasing doses of thrombin. The active site of thrombin was necessary for enhancement, as the serine esterase inhibitor di-isopropyl-fluorophosphate (DIP) and hirudin inhibited the synergy observed with LPS and thrombin. Prothrombin and Factor Xa also enhanced IL-1 production, although not to the same extent as thrombin. Factor Xa-like activity was demonstrated on the surface of LPS-stimulated macrophages. Both the Xa-like activity and IL-1 generated by LPS-stimulated cells were inhibited by heparin. Heparin with a high affinity for antithrombin III (anti-coagulant heparin; HAH) inhibited IL-1 generation, whereas low-affinity heparin (non-anticoagulant; LAH) had no effect. We show that proteases of the extrinsic coagulation cascade enhance IL-1 generation and propose that a Factor Xa-like activity present in activated macrophages, together with thrombin, may be important in IL-1 processing.

Animals↗

PubMed1989

Enhanced interferon-gamma (IFN) production by lymph node cells from autoimmune (MRL/1, MRL/n) mice.

Interferons (IFNs) have been implicated in the aetiopathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). The ability of unstimulated and Sepharose-bound concanavalin A (ConA) stimulated spleen or lymph node (LN) cells from normal CBA/T6 mice and histocompatible autoimmune strains (MRL/1, MRL/n) of various ages to produce IFN-gamma was measured. Levels of IFN-gamma produced by ConA-stimulated spleen cells from CBA/T6, MRL/1 and MRL/n mice at 6 weeks of age were not statistically different (mean +/- SEM: 786 +/- 182, 1147 +/- 282, 1024 +/- 146 IU/ml, respectively). At this age only stimulated LN cells from MRL/l mice produced detectable IFN-gamma (538 +/- 44 IU/ml) and these levels remained constant up to 6 months. IFN production by stimulated LN cells from young MRL/n mice (66 +/- 21 IU/ml at 3 months, 44 +/- 41 IU/ml at 6 months) increased at 1 year (463 +/- 97 IU/ml) corresponding to the age of disease onset. The failure of stimulated CBA/T6 LN cells to produce IFN-gamma was not due to cell-cell suppression, defective IL-2 production or the generation of soluble inhibitors. Stimulated LN cells from other normal inbred (C57Bl/6, Balb/c, A/J) outbred and FI hybrid mouse strains (Swiss, [Swiss x Balb/c] F1, (CBA/T65 x C57Bl/6]FI) produced undetectable or low levels of IFN compared to MRL/1 and MRL/n mice. These results show that autoimmune mouse LNs generate more IFN compared to normal controls and that the increase in IFN levels (at least in MRL/n) corresponds to the age of disease onset.

Animals↗

PubMed1988

Macrophage procoagulant-inducing factor. In vivo properties and chemotactic activity for phagocytic cells.

Murine macrophage procoagulant-inducing factor (MPIF) is a lymphokine with chemical properties distinct from a number of well-characterized cytokines. MPIF induces procoagulant activity on the surface of macrophages and thus may play a central role in the expression of cell-mediated immunity. Highly enriched MPIF-alpha and -beta, separated by virtue of their basic isoelectric point and affinity for heparin, induced local induration and fibrin deposition and cellular infiltration similar to that observed in delayed type hypersensitivity reactions, when injected intradermally. Margination with of polymorphonuclear leukocytes (PMN) along the endothelium as well as increased PMN infiltration was evident after 4 h. In contrast to other inflammatory mediators (e.g., C5a, IL-1) reactivity was sustained, with greater numbers of mononuclear cells apparent 24 h after skin testing. Changes in the dermis were evident 4 h after MPIF injection with increased numbers of cells near areas where spaces in the collagen bundles had formed. Dermal thickening was evident after 24 h and collagen fiber structure was disrupted. Extravascular fibrinogen/fibrin was most prominent 24 h after testing. LPS, which induces macrophage procoagulant activity in vitro, did not induce the histopathologic changes evident with MPIF. MPIF was chemotactic for PMN and macrophages in vitro. Chemotactic activity was heat-labile and not due to C5a. Migration was dependent on a concentration gradient, as determined by checkerboard analysis, indicating that MPIF promoted chemotaxis rather than chemokinesis. Experiments reported here suggest that MPIF is an important mediator of fibrin deposition and cellular infiltration characteristic of cell-mediated immune response.

Animals↗

PubMed1988

Recombinant IFN-gamma synergizes with lipopolysaccharide to induce macrophage membrane procoagulants.

Fibrin deposition is an important histopathologic feature of inflammation and is mediated, in part, by monocyte/macrophage procoagulants. rIFN gamma acted in synergy with suboptimal levels of bacterial LPS by priming thioglycollate-induced mouse peritoneal exudate cells (TG-PEC) to express high levels of surface procoagulant. TFN-alpha beta, TFN-alpha, IL-1, either alone or in combination with LPS or IFN-gamma, had no effect on macrophage procoagulant activity expression. In contrast to the dramatic increases of macrophage procoagulant activity induced by IFN-gamma/LPS, on exudate macrophages, normal peritoneal macrophages, or peripheral blood monocytes were unresponsive suggesting that the state of activation of the macrophage determines reactivity. IFN-gamma induced a Factor VIIa-like activity detected only after cell disruption. Synergy between LPS and IFN-gamma-induced procoagulants may occur as the result of the assembly of the thromboplastin (induced by LPS), Factor VII/VIIa complex on the macrophage surface. RNA synthesis was required for procoagulant induction. Procoagulant expression may, as for other cytokines involved in inflammatory responses, be regulated by short lived repressor proteins as low dose cycloheximide superinduced procoagulant responses to both LPS and IFN-gamma and caused the extracellular expression of procoagulant in response to IFN-gamma. This study suggests an important role for IFN-gamma in the assembly of components of the extrinsic coagulant cascade on the macrophage surface. The synergy between IFN-gamma and LPS may moderate macrophage-initiated fibrin deposition characteristic of inflammatory responses.

Animals↗

PubMed1988

High endothelial venule morphology and function are inducible in germ-free mice: a possible role for interferon-gamma.

Cytokines may facilitate lymphocyte traffic by modulating HEV structure and lymphocyte binding function in accordance with local tissue requirements. This study investigated whether the morphology of HEVs and their lymphocyte binding ligand are altered following antigenic challenge and evaluated the role of IFN-gamma in the induction of such changes. The morphology and lymphocyte binding function of mesenteric LN HEVs of GFM exposed to environmental pathogens were compared to those from GFM and conventional mice. Lymph nodes from all mice had microscopically identifiable HEVs. The morphology of HEVs from GFM was not uniform; many HEVs contained flat endothelial cells with sparse cytoplasm and prominent interendothelial gaps. The number of lymphocytes within the lumen and the HEV wall was low. In contrast, HEVs from GFME and conventional mice were characterized by cuboidal endothelial cells with plentiful cytoplasm and large numbers of lymphocytes in the vessel wall and lumen. There was no delineation of interendothelial cell borders. Lymphocyte binding to HEVs of lymph node sections from GFM was reduced (mean +/- SEM: 1.08 +/- 0.15) compared to that of conventional mice (1.91 +/- 0.20), P less than 0.003. GFME had augmented lymphocyte binding (2.23 +/- 0.26) to levels comparable with those of conventional mice. GFM injected intraperitoneally with IFN-gamma, IFN-alpha beta, or diluent resulted in minor changes in HEV morphology. By contrast, lymphocyte binding to HEV of GFM was more than doubled by the injection of IFN-gamma (1.95 +/- 0.25), P less than 0.01, but not IFN-alpha beta (0.54 +/- 0.07) or the relevant diluent controls (0.89 +/- 0.11, 0.56 +/- 0.06, respectively). It appears that the HEV binding ligand is inducible, and its expression is regulated by at least one immunomodulator, IFN-gamma. Although short-term exposure of HEVs to IFN-gamma influenced HEV function it caused only minor changes in morphology.

Animals↗

PubMed1988

Procoagulant induction by human lymphokine and interferon gamma/PMA on a myelomonocytic cell line, RC2a.

The human myelomonocytic cell line RC2a expressed procoagulant activity following stimulation with human lymphokine (LK) prepared by stimulating peripheral blood mononuclear cells with either mitogens (Concanavalin A, phytohaemagglutinin or antigen (tuberculin). Induction was rapid, with optimal activity observed between 6 and 8 h, was decreased in cultures containing serum and was not inhibited by actinomycin D or cycloheximide. The LK activity was not inhibited by an anti-interferon gamma (IFN gamma) antibody. IFN gamma and phorbol myristate acetate (PMA) had no activity but acted in synergy to induce procoagulant expression; interferon alpha plus PMA were without effect. In contrast to the LK-induced response, procoagulant induced by IFN gamma/PMA was not detected for up to 8 h and steadily rose over 24-48 h culture and was dependent on new protein and RNA synthesis. Bacterial lipopolysaccharide, a potent inducer of thromboplastin on normal human monocytes, failed, either alone or in combination with LK, IFN gamma, PMA or IFN gamma plus PMA, to activate procoagulant expression on RC2a cells. Both LK and IFN gamma/PMA-induced procoagulant had properties of thromboplastin expressed both intracellularly and on intact, viable cells. This study shows that RC2a cells are responsive to factors produced as the result of an activated cell-mediated immune response which may therefore contribute to the coagulopathies common to this form of malignancy.

Blood Coagulation Factors↗

PubMed1987

Hypothesis: lymphocyte trafficking in inflammatory rheumatic disease. A role for receptor mediated homing.

The entry of lymphocytes into normal lymph nodes occurs principally after their passage across postcapillary high endothelial venules. Nonlymphoidal tissues do not normally express high endothelial venules; however, in inflammatory rheumatic diseases associated with prominent lymphocyte accumulation (e.g., rheumatoid synovium), high endothelial venule-like structures are found and may facilitate local lymphocyte traffic. In mouse and man there are lymphocyte surface membrane homing receptors which determine lymphocyte binding to acceptor sites on lymph node high endothelial venules. The migration of lymphocytes from the circulation into inflamed nonlymphoidal tissues may be controlled by similar homing receptor mediated mechanisms.

Adrenal Cortex Hormones↗

PubMed1986

Characterization and purification of mouse macrophage procoagulant-inducing factor.

Macrophage procoagulant-inducing factor (MPIF) is a product of mouse Lyt-1+2- cells that induces macrophage procoagulant activity (MPCA) on mouse peritoneal exudate cells or on the macrophage-like tumor cell line WEHI-265. Supernatants from Sepharose-bound concanavalin A-stimulated cells were fractionated by using DEAE-Sephacel, heparin-Sepharose, and isoelectric focusing. This procedure resolved three different MPIF: MPIF alpha (pI 8.5), MPIF beta (pI 8.8 to 9.2), and MPIF gamma (pI 5 to 5.5). MPIF alpha and beta were small molecules (approximately 14 kD and 20 to 25 kD) as determined by gel filtration on Sephadex G200 and Biogel P100. MPIF beta was sharply resolved as a peak eluting after lysozyme by gel filtration on HPLC columns I-150 and I-125, although SDS-PAGE of the HPLC-enriched material resolved two well-defined bands of 70 and 120 kD and some poorly defined material of 14 kD. Silver staining failed to detect components of MPIF alpha after SDS-PAGE. MPIF gamma activity was associated with material that separated over a broad range (20 to 60 kD and 60 to 200 kD), possibly due to aggregation with other components of the supernatants. Crude supernatants were stable to heating at 56 degrees C for 30 min and pH 2 treatment, although more highly enriched fractions were unstable to these treatments. Heating at 90 degrees C for 5 min totally destroyed MPIF activity. The properties of the two basic MPIF differ from other lymphokines known to affect macrophage function, e.g., colony-stimulating factor, migration-inhibition factor, interferon-gamma, and interleukin 1.

Animals↗