Suppression of natural killer and lymphocyte functions associated with carcinogen-induced premalignant hyperplastic nodules in rat liver.

The effects of chemical carcinogenesis to produce premalignant hyperplastic nodules in rat liver on concomitant immune function were studied. Induction of hyperplastic nodules in Fischer rats was accomplished using a combined regimen of diethylnitrosamine, 2-acetylaminofluorene, and partial hepatectomy. Hyperplastic nodules were detected in carcinogen-treated rats from 5 to 23 weeks as confirmed by gross pathology, histopathology, and significantly elevated liver gamma-glutamyltransferase activity. Suppression of natural killer activity of either peritoneal or peripheral blood lymphoid, but not splenic, cells for YAC-1 target cells occurred during 5 to 20 weeks in carcinogen-treated rats. Spleen and blood lymphocyte mitogenic responses to concanavalin A and pokeweed mitogen were also suppressed at most intervals from 8 through 20 weeks. Control groups given individual carcinogen or partial hepatectomy alone or in dual combination were not suppressed in their immune function and failed to develop hyperplastic foci or changes in liver gamma-glutamyltransferase. Our findings indicate that immunosuppression of natural killer and lymphocyte mitogenic functions occurs for a protracted period concurrently with the development of the premalignant hyperplastic state in rat liver. The data suggest a potential role for immune competency during the onset of malignant neoplasia.

Kinetics of the biosynthesis of complement subcomponent C1q by murine macrophages: LPS, immune complexes, and zymosan alone and in combination with interferon-gamma.

We investigated the effects of bacterial lipopolysaccharide (LPS), immune complexes (IC), and C3b opsonized zymosan (AZ) alone and in combination with interferon-gamma (IFN-gamma) priming on macrophage synthesis and secretion of C1q. Our results indicated that LPS, IC, and AZ alone stimulated C1q mRNA and secretion in the absence of IFN-gamma. The increase in mRNA accumulation was detectable after 3 h, peaked at 6 h and was maintained at constitutive levels for 24 h. There was a corresponding early burst of increased secretion of functional C1q after 3 to 6 h which declined rapidly after 9 to 24 h culture of LPS-stimulated macrophages. Priming of macrophages with IFN-gamma and simultaneous triggering with LPS, IC, or AZ produced additive rather than synergistic increases in C1q mRNA accumulation. These same agents inhibited constitutive secretion of C1q in the absence of IFN-gamma priming as determined by autoradiographic analysis of metabolically radiolabeled secretory C1q. Triggering of IFN-gamma primed macrophages with LPS, IC, or AZ also markedly suppressed the increased rate of C1q secretion induced by IFN-gamma in a dose-related fashion. A corresponding dose-dependent increased accumulation of endogenous C1q in cell lysates was detected by Western blot analysis of macrophages which had been stimulated by LPS, IC, or AZ alone or in combination with IFN-gamma. Our findings indicate that LPS as well as FcR and C3bR triggering agents stimulate early and sustained C1q synthesis accompanied by an early and short-lived burst of C1q secretion which rapidly diminished and results in an increased intracellular accumulation of C1q due to ongoing synthesis. IFN-gamma appeared to further amplify the same kinetics of increased C1q mRNA accumulation and decreased extracellular accumulation mediated by LPS, IC, and ZM. Our results suggest that LPS, IC, and AZ alone or in combination with IFN-gamma stimulate early C1q production to modulate macrophage effector functions followed by an inhibition of C1q secretion when the activation process has been culminated.

Microassay for photometric quantitation of macrophage mediated tumor cytotoxicity using an automated densitometer.

A simple and reproducible microassay for the quantitation of macrophage mediated cytotoxicity is described. The method is based on the measurement of absorbance at 630 nm of residual Giemsa stained target cells and effector macrophages using an automated densitometer. Applying this novel method, it was possible to demonstrate time dependent growth characteristics of C3H/MCA and BHK/Py target cell lines. Using C3HeB/FeJ or C3H/HeJ murine effector macrophages and syngeneic transformed fibroblast target cells (C3H/MCA or 3T12), the method was further applied to demonstrate: (1) dose related activation of macrophages by lipopolysaccharide (LPS) and by macrophage activating factor (MAF); (2) synergistic augmentation of MAF-mediated macrophage cytotoxicity by LPS; (3) unresponsiveness of C3H/HeJ macrophages to LPS; and (4) increased cytotoxicity with increasing effector: target cell ratios. Guinea pig peritoneal macrophages were also shown to produce enhanced LPS or MAF-mediated cytotoxicity for C3H/MCA or BHK/Py target cells. The novel method was shown to compare favorably with results obtained by cytotoxic release of [3H]thymidine from prelabeled target cells. The advantages of the method are: (1) the elimination of the need for radioactive materials; (2) the ability to perform quantitation directly in microtiter plates; (3) the relative ease and rapidity in which experiments may be performed and quantitated; (4) its sensitivity and reproducibility; and (5) the ability to simultaneously quantitate and observe the biological events either microscopically or macroscopically.

Binding characteristics of fluoresceinated Lotus tetragonolobus fucolectin to macrophage populations which are either responsive or refractory to activation by lymphokine.

Fucose binding protein (FBP) from Lotus tetragonolobus seed was studied by fluorescence microscopy for its binding characteristics to various guinea pig peritoneal macrophage populations. Fluoresceinated FBP (FITC-FBP) was bound optimally at 22 degrees C in a punctate distribution and was internalized at 37 degrees C. Binding of FBP to macrophages was reversed specifically by the competitive sugar L-fucose, and not by D-fucose, L-rhamnose, or D-galactose. FBP was bound with greater frequency and intensity to 3-day oil-elicited peritoneal macrophages which are responsive to migration inhibition by FBP and migration inhibitory factor (MIF) than to resident or 7-day inflammatory macrophages which are unresponsive to activation by the same effectors. Competition for visual binding of FITC-FBP to macrophages was demonstrated by preincubation of cells with unlabeled FBP or MIF. Competition of FITC-FBP binding by MIF was reversed by L-fucose. These results indicate that FBP binds preferentially, with greater frequency and intensity, to macrophage subpopulations which are responsive to MIF than to MIF-refractory macrophages. The data further supports the existence of a common receptor site for MIF and FBP on the macrophage membrane which involves fucosyl determinants.

Characterization of Lotus tetragonolobus fucolectin components for differences in hemagglutinating and macrophage activating activities.

The fucose binding proteins (FBP) extracted from Lotus tetragonolobus seeds were isolated by affinity chromatography and compared with affinity purified commercial preparations for physical, antigenic, and biological properties. All preparations contained three protein components as determined by polyacrylamide gel electrophoresis, each with a subunit molecular weight of approximately 27 Kd. FBP preparations were also found to be antigenically identical by immunodiffusion analysis and possessed similar biological activities for hemagglutination of group 0 erythrocytes and macrophage activation in the migration inhibition assay. A reversible temperature dependent hemagglutination characteristic was found; FBP agglutinated erythrocytes at 4 degrees and 22 degrees C but not at 37 degrees C, which was reversed by decreasing the incubation temperature from 37 degrees C to 22 degrees C. Temperature dependent binding of FBP for macrophages was also demonstrated. Adsorption of crude FBP by group 0 erythrocytes preferentially removed hemagglutinin without loss of macrophage activating properties. Similarly adsorption of FBP with macrophages preferentially removed macrophage activating component. Separation of the lectin components by DEAE cellulose chromatography yielded two major fractions: a potent hemagglutinin with weak macrophage activating properties and a potent macrophage activator with weak hemagglutinating activity. Separation of the crude lectin by ultrafiltration indicated that the macrophage activating component exists in a highly aggregated form which may determine its macrophage activating properties. Our results indicate that L. tetragonolobus consists of two distinct classes of components which correspond to tetrameric glycoproteins of 118-120 Kd with potent temperature dependent hemagglutinating activity and a highly aggregated dimeric component of 58 Kd with macrophage activating properties.

Relationship between murine macrophage Fc receptor-mediated phagocytic function and competency for activation for non-specific tumor cytotoxicity.

The relationship between Fc receptor (FcR) function and activation of murine macrophage populations for non-specific tumor cytotoxicity was studied. Oil-elicited inflammatory peritoneal macrophages (PM phi) from C3HeB/FeJ mice had higher FcR function upon harvest than resident PM phi from the same strain or elicited PM phi from genetically deficient C3H/HeJ mice. C3HeB/FeJ inflammatory PM phi were uniformly responsive to activation by MAF and the complement activators: LPS, Poly I:C, cobra venom factor (CVF) and zymosan for tumoricidal activity. Resident cells from the same strain and C3H/HeJ-elicited PM phi were uniformly unresponsive to the same activators. In vitro culture of C3HeB/FeJ resident PM phi with fetal bovine serum for 24-48 h produced unregulation of FcR function which coincided with a conversion from an unresponsive to a responsive state for tumoricidal activity. Reconstitution of the FcR function of C3H/HeJ-elicited PM phi during 24-48 h culture with lymphokine or Poly I:C also coincided with the restoration of responsiveness to activation by LPS, CVF, and zymosan for tumor cytotoxicity. Thus, the consistent temporal relationship between upregulated FcR function and the capacity of macrophages to respond to activation for non-specific tumoricidal activity may be more than coincidental. Preincubation of responsive C3HeB/FeJ-elicited PM phi with insoluble immune complex or heat-aggregated IgG was shown to blockade FcR-mediated phagocytosis and to abrogate LPS-mediated tumoricidal activity. Interestingly, FcR blockade by IgG-opsonized sheep erythrocyte conjugates selectively inhibited activation by MAF, LPS, and Poly I:C, but had no inhibitory effect on activation by CVF or zymosan. Similar blockade of C3b receptors produced an identical pattern of selective inhibition of activation. This selective inhibition of non-specific tumoricidal activity by FcR/C3bR blockade suggests the existence of two pathways for antibody-independent activation of macrophages.

Inhibitor of C1q secretion suppresses the macrophage response to lipid A for nitric oxide but not for TNF production: evidence for a role of C1q in autocrine binding of TNF.

Studies were designed to further define the modulatory role of complement subcomponent C1q in macrophage activation by Lipid A to mediate production of TNF and cytotoxic nitric oxide (NO). Pretreatment of macrophages for 24 h with 2.5 mM 3,4,dehydro-D,L-proline (DHP), an inhibitor of C1q secretion, suppressed their response to Lipid A activation for cytotoxicity of P815 tumor targets which correlated with a corresponding decrease in NO production. In contrast, DHP-pretreated macrophages displayed an increase in the release of TNF in response to Lipid A as compared to untreated controls. Time kinetic studies indicated that DHP-pretreated macrophages produced higher sustained levels of TNF activity during 1 to 24 h culture with Lipid A than did untreated control macrophages. This was confirmed by increased TNF mRNA expression in response to Lipid A by DHP-treated cells. DHP-pretreated macrophages had reduced levels of cell surface C1q as determined by cytofluorometric analysis of the binding of FITC-labeled anti-C1q, F(ab')2. Macrophages were also found to have reduced binding capacity for phycoerythrin-labeled rTNF (PE-TNF) by cytofluorometric analysis following DHP treatment. Exposure of DHP-pretreated macrophage to soluble C1q at 4 degrees C restored their reduced binding of PE-TNF. C1q was confirmed to bind to macrophages at 4 degrees C as detected by FITC anti-C1q, F(ab')2 and such C1q binding promoted a corresponding increased binding of PE-TNF. Macrophages which were plated over immobilized C1q were also markedly enhanced in their binding of PE-TNF probe. Our results indicate that the inhibition of macrophage secretion of C1q by DHP pretreatment, was accompanied by an increased TNF mRNA expression and release with a decrease in NO generation following Lipid A activation. Since TNF binding to DHP-treated macrophages was reconstituted by the binding of exogenous C1q to the cells, it appears that C1q may be involved in the modulation of autocrine binding of TNF for subsequent generation of cytotoxic NO.

Implementing and evaluating a system of generic infection precautions: body substance isolation.

Body substance isolation (BSI) is a system of infection precautions intended to reduce nosocomial transmission of infectious agents among patients and to reduce the risk of transmission of hepatitis B virus, human immunodeficiency virus, and other infectious agents to health care personnel. Harborview Medical Center in Seattle, Wash., was the first facility in the United States to implement the BSI system. Between 1984 and 1988 a systematic evaluation of the implementation process was conducted and the effects of BSI on appropriate glove use by hospital personnel and on the incidence of nosocomial colonization and infection by sentinel organisms was measured. Results of the evaluation showed (1) significant increments in knowledge of infection control procedures and practices as measured by comparing written examination responses before and after training sessions, (2) significant increases in appropriate glove use as determined by direct observation of hospital employees for 18 months, and (3) significant reductions in nosocomial colonization and infection caused by sentinel microorganisms during the period from 1984 to 1988.

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.

Activation of mouse macrophages for migration inhibition and for tumor cytotoxicity is mediated by interferon-gamma priming and triggering by various agents.

The requirements for activation of C3HeB/FeJ mouse peritoneal macrophages to mediate migration inhibition from capillary tubes was compared with those conditions prerequisite for nonspecific tumor cytotoxicity. Both in vitro assays for macrophage activation were found to require a two-stage process that involved priming by murine recombinant interferon-gamma (IFN-gamma) and triggering by subactivating concentrations of bacterial lipopolysaccharide (LPS), Lipid A, Polyinosinic-polycytidylic acid (poly I:C), or cobra venom factor (CVF). A dose-related increase in both migration inhibition and tumor cytotoxicity was shown with increasing concentrations of IFN-gamma (3.0-50.0 U/ml) in synergistic combination with an LPS trigger. IFN-gamma alone produced low levels of migration inhibition or tumor cytotoxicity, only at higher concentrations, that was not attributable to LPS contamination. The concentrations of the various agents required for direct activation or triggering of IFN-gamma-primed macrophages were approximately 2- to 10-fold greater for migration inhibition than for tumor cytotoxicity. Our results indicate that the two-signal process of priming and triggering for mediating mouse macrophage nonspecific tumoricidal activity is also operative in migration inhibition from capillary tubes. Thus, under defined conditions with purified lymphokines, the migration inhibition assay appears to be a reliable alternate in vitro correlate of macrophage activation by IFN-gamma.

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, epsilon-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.

Kinetics of the biosynthesis of complement subcomponent C1q by murine macrophages: effects of stimulation by interferon-gamma.

The effects of interferon-gamma (IFN-gamma) on the kinetics of biosynthesis of complement subcomponent C1q by mouse inflammatory peritoneal macrophages were determined. Stimulation of macrophages with various concentrations of IFN-gamma produced a dose-dependent increase in C1q mRNA accumulation which was detected as early as 3 h and sustained through 24 h, as determined by Northern blot analysis. A corresponding early increase in the extracellular accumulation of functional C1q was detected in culture supernatants after 3-9 h stimulation of macrophages with IFN-gamma that was sustained for 24-48 h as determined by a complement hemolytic assay. Autoradiographic analysis of [35S]methionine-labeled secretory C1q confirmed the protracted dose-dependent secretion of C1q by IFN-gamma stimulated macrophages during 24-48 h of culture. Western blot analysis of macrophage lysates indicated no significant changes in endogenous C1q levels following stimulation with IFN-gamma either after 3-9 h or 24-48 h when both C1q mRNA and extracellular accumulation were at their peak. Our results indicate that IFN-gamma promotes early and protracted mRNA accumulation and secretion of C1q by macrophages without intracellular accumulation, presumably due to the rapid rate of secretion of newly synthesized C1q. It is apparent that priming of macrophages with IFN-gamma provides a rapid and abundant source of secretory C1q for potential interaction with various macrophage triggering agents which also bind C1q.

Inhibitors of C1q biosynthesis suppress activation of murine macrophages for both antibody-independent and antibody-dependent tumor cytotoxicity.

The inhibitors of C1q biosynthesis and secretion, 3,4-dehydro-DL-proline (DHP) and 2,2'-dipyridyl, were previously shown to suppress murine macrophage FcR-dependent phagocytosis and cytolysis of IgG-opsonized RBC targets. Inasmuch as non-antibody macrophage activators also bind C1q to initiate C1 activation, we determined the effects of these same inhibitors of C1q biosynthesis on activation of macrophages for antibody-independent, nonspecific tumor cytotoxicity by lipid A and a variety of other non-antibody activators. Preexposure of mouse inflammatory peritoneal macrophages to either DHP (0.5 to 2.5 mM) or 2,2'-dipyridyl (0.1 to 0.3 mM) for 24 h produced a dose-related suppression of their response to activation by lipid A to mediate tumor cytotoxicity of L1210 mouse leukemia targets. Inhibition of C1q secretion by DHP-treated macrophages was confirmed both by a complement hemolytic assay and by autoradiographic analysis of [35S]methionine-labeled culture supernatants. DHP-treated macrophages were inhibited in their response to direct activation and triggering of IFN-gamma-primed macrophages by lipid A, Poly I:C, and cobra venom factor for tumor cytotoxicity. DHP inhibited macrophage activation for antibody-dependent cellular cytotoxicity of L1210 tumor targets mediated by antitumor target IgG. The addition of exogenous purified C1q (2 micrograms/ml) to macrophages after DHP treatment, reconstituted their response to activation for both antibody-independent and antibody-dependent tumor cytotoxicity. Our results indicate that C1q synthesis and secretion by effector macrophages is a prerequisite for the initiation of their activation by both immune complex and by non-antibody agents that also bind C1q. It now appears that macrophage-derived C1q may act as an auxiliary amplification signal for autocrine-like modulation of the initiation of macrophage activation by both the antibody-dependent and independent pathways.

Mechanistic differences between migration inhibitory factor (MIF) and IFN-gamma for macrophage activation. MIF and IFN-gamma synergize with lipid A to mediate migration inhibition but only IFN-gamma induces production of TNF-alpha and nitric oxide.

Previously we found that murine macrophage migration inhibition (MMI) was mediated by IFN-gamma-priming and lipid A triggering. With the recent availability of human recombinant migration inhibitory factor (MIF), which is distinctly different from IFN-gamma and other cytokines, we have now attempted to explore possible mechanistic differences between IFN-gamma and MIF to mediate MMI. Neither MIF not IFN-gamma were active alone, but effectively primed murine inflammatory macrophages for subsequent triggering by lipid A to mediate MMI. A specific neutralizing antibody for rMIF abrogated MMI mediated only by MIF and not by IFN-gamma-primed macrophages. Distinct differences were also found between the mechanisms by which MIF and IFN-gamma synergized with lipid A for activation in that IFN-gamma-primed and lipid A triggered macrophages produced TNF and nitric oxide (NO), whereas MIF-primed cells did not. Macrophages primed with IFN-gamma and triggered by rTNF were inhibited in their migration, whereas MIF failed to synergize with rTNF for MMI. An inhibitor of NO production NG-monomethyl-L-arginine inhibited MMI mediated by higher activating concentrations of lipid A and by IFN-gamma-primed and lipid A triggered macrophages, but had no effect on MIF-primed cells in concert with lipid A for increased expression of both TNF-alpha mRNA and NO synthase mRNA. Taken together, our results indicate that both MIF and IFN-gamma prime macrophages to synergize with lipid A to mediate MMI but by different mechanisms. The activation process by IFN-gamma to mediate migration inhibition appears to resemble requirements for rendering macrophages tumor cytotoxic in the production of TNF for autocrine-mediated NO generation by primed macrophages. In contrast, MIF-mediated MMI was independent of requirements for either TNF or NO production.

Reconstitution of murine resident peritoneal macrophages for antibody-dependent cellular cytotoxicity by homologous serum Clq.

Mouse resident peritoneal macrophages (PM) were reconstituted in their response to activation for antibody-dependent cellular cytotoxicity (ADCC) for sheep erythrocyte targets (SRBC) by subhemolytic dilutions of homologous or autologous sera. ADCC-responsive inflammatory PM were largely unaffected in their activation by exogenous serum. Augmentation of resident PM for ADCC by homologous serum was correlated with the complement-activating potential of the mouse monoclonal anti-SRBC IgG isotype in that serum augmented IgG gamma 2a greater than IgG gamma 2b much greater than IgG gamma 1. The active component of mouse serum was heat-labile at 56 degrees C for 30 min and was present in both C5-deficient AKR and C5-sufficient homologous C3H mouse sera. Western blot analysis of the cell lysates for Clq confirmed that oil-elicited and thioglycollate-elicited inflammatory PM had greater levels of endogenous Clq than did resident PM which correlated with their innate responsiveness for ADCC activation. Depletion of Clq from serum by immunoprecipitation with IgG antibody to Clq or by ion exchange chromatography removed the active reconstituting activity for ADCC. Purified mouse Clq (0.4 microgram) partially replenished the ADCC augmenting activity of Clq-depleted AKR mouse serum. SRBC targets preopsonized with IgG gamma 2a and purified mouse Clq (0.075-5.0 microgram/ml) fully reconstituted the ADCC response of resident PM similar to homologous serum indicating that the major active component of serum was Clq. Thus resident PM with low endogenous levels of Clq were reconstituted for ADCC by the addition of exogenous Clq, whereas inflammatory PM with sufficiently high endogenous levels of Clq were not further enhanced by exogenous Clq. Our findings indicate that Clq may provide an essential second signal in concert with Fc receptor binding of IgG to initiate ADCC activation of macrophages.

Staphylococcal exotoxins stimulate nitric oxide-dependent murine macrophage tumoricidal activity.

The staphylococcal exotoxins toxic shock syndrome toxin 1 (TSST-1) and enterotoxin B were tested for their ability to stimulate murine peritoneal macrophages (PM) for tumoricidal activity. Both toxins were found to stimulate oil-elicited, gamma interferon-primed PM monolayers to kill nonadherent P815 tumor targets. The mechanism of killing of toxin-stimulated tumoricidal activity involved the production of nitric oxide, as nitrite could be demonstrated in culture fluids, and NG-monomethyl-L-arginine, an inhibitor of nitric oxide production, abrogated toxin-stimulated tumoricidal activity. TSST-1 stimulated the secretion of tumor necrosis factor by PM monolayers in the presence and absence of gamma interferon. The mechanism of toxin-stimulated tumoricidal activity was also determined to be independent of the production of reactive oxygen intermediates in that TSST-1 failed to stimulate H2O2 production by PM. These results demonstrate that the staphylococcal exotoxins are capable of stimulating macrophage production of nitric oxide for tumor cytotoxicity and suggest that the nitric oxide thus produced may subsequently play a role in the pathogenesis of the diseases caused by these toxins.

Evidence for endogenous C1q modulates TNF-alpha receptor synthesis and autocrine binding of TNF-alpha associated with lipid A activation of murine macrophages for nitric oxide production.

The role of endogenously synthesized complement subcomponent C1q on autocrine binding of tumor necrosis factors (TNF-alpha) and on TNF-alpha receptor (TNF-R) mRNA synthesis by mouse macrophages was investigated. Activation of C3H mouse peritoneal macrophages (C3H-PM phi) by Lipid A induced TNF-alpha and nitric oxide (NO) to kill tumor targets. Such activation also increased macrophage-endogenous C1q synthesis and secretion in a dose-dependent fashion. Antibody for C1q markedly inhibited C3H-PM phi NO production in response to Lipid A, but had no effect on TNF-alpha production. C3H-PM phi treated with C1q or Lipid A displayed increased TNF-R mRNA synthesis and in combination with Lipid A and anti-C1q antibody inhibited TNF-R and nitric oxide synthase (NOS) mRNA synthesis compared with Lipid A only, but had no effect on TNF mRNA synthesis. In vitro treatment of C3H-PM phi with C1q also increased TNF-alpha binding to their surfaces. Taken together, the data indicate that endogenously synthesized C1q is operative in promoting TNF-R mRNA synthesis and resultant autocrine binding of TNF-alpha for induction of NOS in the process of NO-mediated tumor cytotoxicity by Lipid A-activated macrophages.

Complement subcomponent C1q modulation of TNF-alpha binding to L929 cells for enhanced TNF-mediated cytotoxicity.

Complement subcomponent C1q has been recently implicated in the modulation of autocrine binding of TNF-alpha to murine macrophages for induction of nitric oxide synthase. In the present study, the putative role of C1q in increasing TNF-alpha binding to L929 cells to mediate cytotoxicity was explored. TNF-sensitive L929 cells (L929-S) had higher total endogenous cellular and surface C1q levels and bound correspondingly more phycoerythrin-labelled rTNF-alpha (PE-TNF) than did a TNF-resistant L929 variant (L929-R). Pretreatment of L929-S with soluble C1q increased their sensitivity to TNF-mediated cytotoxicity coincident with increased binding of PE-TNF, but similar treatment of L929-R had no effect. Pretreatment of L929-S with an inhibitor of C1q secretion, 3,4 dehydro-D,L-proline (DHP), resulted in a decrease in their TNF-mediated cytotoxicity, as well as reduced binding of PE-TNF. Subsequent exposure of DHP-treated L929-S with exogenous soluble C1q restored their TNF-mediated cytotoxicity and binding of PE-TNF. These results provide evidence for the modulation of TNF-alpha binding to TNF sensitive tumour targets L929 by either endogenously synthesized or exogenously added C1q to promote TNF-mediated cytotoxicity by mechanisms which remain to be elucidated.