HIF-1 expression in healing wounds: HIF-1alpha induction in primary inflammatory cells by TNF-alpha.

The expression of the hypoxia-responsive transcription factor hypoxia-inducible factor (HIF)-1 during acute inflammation was investigated in experimental wounds. HIF-1alpha mRNA was maximally expressed in wound cells 6 h after injury. HIF-1alpha protein was detectable in wound cells 1 and 5 days after injury. Cells from 1-day-old wounds were not hypoxic, as determined by lack of pimonidazole hydrochloride adduct formation. Tumor necrosis factor (TNF)-alpha, but not interleukin-1beta, increased the HIF-1alpha protein content of cells isolated 1 and 5 days after injury, and also of glycogen-elicited peritoneal cells, but not HIF-1alpha mRNA. HIF-1alpha did not accumulate in TNF-alpha-treated HeLa, NIH/3T3, NR8383, or RAW 264.7 cells. Nitric oxide from S-nitrosoglutathione did not induce HIF-1alpha accumulation or modulate the response to TNF-alpha. TNF-alpha did not increase oxygen consumption or result in the production of reactive oxygen intermediates by day 1 wound cells. Vascular endothelial growth factor mRNA in wound cells peaked 24 h after wounding. HIF-1 expression in early wounds may contribute to the regulation of inducible nitric oxide synthase and vascular endothelial growth factor, two HIF-1-responsive genes intimately related to the process of repair.

Receptor-mediated phagocytosis of rat macrophages is regulated differentially for opsonized particles and non-opsonized particles containing beta-glucan.

Experiments were conducted to test the hypothesis that opsonic and non-opsonic phagocytic capacities are differentially regulated by resting and wound-derived macrophages. Furthermore, the phagocytosis of non-opsonized zymosan and beta-glucan particles was quantified to determine whether cells differentially regulate non-opsonic lectinophagocytosis in accordance with the carbohydrate composition of the ligand. In that regard, wound macrophages exhibited profound differential regulation in lectinophagocytosis with a seven-fold increase in phagocytosis of beta-glucan particles following overnight culture but with a relatively modest increase in internalization of mannan-containing zymosan. Cultured peritoneal macrophages increased uptake of both particles similarly. Upon activation with interferon-gamma/lipopolysaccharide (IFN-gamma/LPS), wound macrophages selectively suppressed beta-glucan ingestion, while phagocytosis of zymosan particles was unaffected. Lectinophagocytosis was decreased in activated peritoneal macrophages regardless of particle composition and was due in part to a nitric oxide-dependent mechanism which was without a role in regulation of wound macrophage lectinophagocytosis. Overnight culture of wound macrophages suppressed their capacity for opsonic-dependent phagocytosis independently of activation, whereas suppression of phagocytosis by peritoneal macrophages was activation-dependent. Regulation of all three phagocytic pathways was achieved distinctly by peritoneal and wound-derived macrophages, with changes found in the percentage of resident peritoneal macrophages capable of phagocytosis, whereas the phagocytic capacity of wound macrophages was primarily affected by the number of particles ingested by individual cells. Taken together, these findings demonstrate that the differential regulation of phagocytic pathways encompasses the nature of the phagocytic particle, the site from which macrophages are obtained, their response to activating agents and the mechanism through which the cell population alters its phagocytic potential.

Macrophage-induced neutrophil apoptosis.

Macrophages (Mphi) contribute to the resolution of early inflammation by recognizing and ingesting apoptotic polymorphonuclear neutrophils (PMN). In addition, experiments reported here demonstrated that Mphi can actively induce PMN apoptosis. Coculture of cells from 2- or 5-day-old wounds in rats, or of Mphi purified from such preparations, with PMN-rich wound cell populations obtained 1 day after wounding increased PMN apoptosis by >3-fold. Neither resident- nor Proprionibacterium acnes-elicited peritoneal Mphi-induced PMN apoptosis. Apoptosis was not mediated by a soluble factor and required E:T contact. Fixed wound-Mphi and membrane isolates from viable Mphi were as effective as intact cells in inducing PMN apoptosis. Mphi-induced apoptosis was inhibited by peptide Arg-Gly-Asp-Ser, anti-beta3 (CD61) Ab, CD36 peptide, or anti-TNF-alpha Ab. Soluble TNF-alpha did not induce PMN apoptosis. In additional studies, K562 cells (negative for beta3, TNF-alpha, and Fas ligand) transfected to express either alphavbeta3 integrin, an uncleavable membrane form of TNF-alpha, or both were used in cocultures with wound PMN. Only the double transfectants were able to induce PMN apoptosis, an effect inhibited by anti-beta3 (CD61) or anti-TNF-alpha Abs. These results demonstrate that wound Mphi induce PMN apoptosis through a constitutive effector mechanism requiring both intercellular binding through integrin-ligand interactions and membrane-bound TNF-alpha.

Vestigial respiratory burst activity in wound macrophages.

Macrophages from experimental wounds in rats were tested for their capacity to generate reactive oxygen intermediates. Measurements of superoxide and H2O2 release, O-2-dependent lucigenin chemiluminescence, oxygen consumption, hexose monophosphate shunt flux, and NADPH oxidase activity in cell lysates indicated, at best, the presence of a vestigial respiratory burst response in these cells. The inability of wound cells to release O-2 was not rekindled by priming with endotoxin or interferon-gamma in vivo or in vitro. NADPH oxidase activity in a cell-free system demonstrated that wound macrophage membranes, but not their cytosols, were capable of sustaining maximal rates of O-2 production when mixed with their corresponding counterparts from human neutrophils. Immune detection experiments showed wound macrophages to be particularly deficient in the cytosolic component of the NADPH oxidase p47-phox. Addition of recombinant p47-phox to the human neutrophil-cell membrane/wound macrophage cytosol cell-free oxidase assay, however, failed to support O-2 production. Present findings indicate an unexpected deficit of wound macrophages in their capacity to generate reactive oxygen intermediates.

Acyl phosphatase activity of NO-inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH): a potential mechanism for uncoupling glycolysis from ATP generation in NO-producing cells.

Treatment of glyceraldehyde-3-phosphate - dehydrogenase (GA (GAPDH) with the NO donors S-nitrosoglutathione, 3-morpholinosydnonimine or diethylamine NONOate (diethylamine diazeniumdiolate) in vitro, inhibited its dehydrogenase activity and induced its acyl phosphatase activity. NO-producing cells, in turn, exhibited reduced GAPDH activity, increased glycolysis, and decreased ATP content, synthesis and turnover. These cellular alterations could be explained by the uncoupling of glycolytic flux from substrate level phosphorylation by the acyl phosphatase activity of NO-modified GAPDH.

Promotion of neutrophil chemotaxis through differential regulation of beta 1 and beta 2 integrins.

Migration of neutrophils requires sequential adhesive and deadhesive interactions between beta 1 and beta 2 integrins and components of the extracellular matrix. Prompted by reports that describe interaction of soluble beta-glucan with the beta 2 integrin Mac-1, a role for beta-glucan in regulation of integrin-mediated migration was investigated. Neutrophil migration in response to fMLP was assessed using an agarose overlay method with slides precoated with fibronectin (Fn) +/- beta-glucan. On Fn, random migration in excess of directed migration was observed. In contrast, migration on Fn + beta-glucan was directional, with marked diminution of random migration. This conversion of random to directed migration was seen neither when Fn was supplemented with alternative polysaccharides nor when beta-glucan was applied to other components of the extracellular matrix. This effect of beta-glucan was shown to be cation dependent and to be effected by Arg-Gly-Asp-containing peptides consistent with an integrin-mediated event. mAb inhibition studies demonstrate that beta-glucan effects this shift toward directed migration through suppression of migration mediated by Mac-1 and very late Ag 5 and enhancement of very late Ag 3-mediated migration. Adhesion assays suggest that the prochemotactic influence of beta-glucan is due, in part but not entirely, to modulation of PMN adhesion to Fn. In summary, these data support a novel role for beta-glucan in regulation of beta 1- and beta 2-mediated neutrophil migration on Fn.

The effect of surgical wounding on tumour development.

For more than a century, a role for wound healing in the outgrowth of tumours has been implied based on observations in both experimental and clinical studies. Wound healing can be divided into stages of inflammatory, proliferative, repair and remodelling processes. Through proper regulation of activation of epithelial, endothelial and inflammatory cells, platelets and fibroblasts, and the production of growth factors, wounds heal and the various cell types resume their normal function. In tumour growth, similar processes of cell activation and growth factor production are observed. These processes are, however, differently regulated leading to ongoing cellular activation. In recent years, growth factors such as EGF, TGF-alpha and TGF-beta, bFGF, IGF I and II, and PDGF have been identified to play a role in the different stages of wound healing. In addition, some of these factors have now been identified as also being involved in the outgrowth of tumours. In this review, cell types involved in wound healing and tumour growth, as well as the growth factors and cytokines they produce and the role of the extracellular matrix, extensively present in both conditions, are being discussed. A better understanding of the time interval during which the sequelae of events in wound healing occur in relation to the time interval of tumour recurrence may be the basis for defining new therapeutic strategies that can interfere with tumour outgrowth without affecting wound healing processes. These new therapeutic approaches may be of importance especially after surgery or other invasive (diagnostic) procedures in cancer patients.

Molecular and metabolic evidence for the restricted expression of inducible nitric oxide synthase in healing wounds.

Tissue injury initiates a temporally ordered sequence of local cellular and metabolic responses presumably necessary for successful repair. Previous investigations demonstrated that metabolic evidence for nitric oxide synthase (NOS) activity is detectable in wounds only during the initial 48 to 72 hours of the repair process. Present results identify the cell types contributing inducible NOS (iNOS) to experimental wounds in rats. iNOS antigen was expressed in most macrophages present in wounds 6 to 24 hours after injury, and these cells exhibited NAPDH diaphorase and NOS activity. Polymorphonuclear leukocytes contained little iNOS antigen and no NADPH diaphorase activity and were minimally able to convert L-arginine to L-citrulline. The frequency of iNOS-positive macrophages declined on days 3 and 5 after wounding. By day 10, most macrophages in the wound were negative for iNOS. These cells, however, acquired iNOS antigen and activity in culture. Wound fluids, but not normal rat serum, suppressed the induction of iNOS during culture. Findings indicate that the expression of iNOS in healing wounds is restricted to macrophages present during the early phases of repair and that components of wound fluid suppress the induction of iNOS in macrophages in late wounds. Polymorphonuclear leukocytes contribute little iNOS activity to the healing wound.

Macrophage phagocytosis of wound neutrophils.

Resolution of acute inflammation is thought to require the recognition and phagocytosis of apoptotic neutrophils (PMN) through receptor-ligand interactions with macrophages (Mphi). This hypothesis was tested in rat wounds by quantifying apoptosis in freshly harvested and aged-in-culture PMN taken from wounds 1-3 days after injury and by using these wound PMN as phagocytic targets for wound, immune-activated peritoneal, and resident peritoneal Mphi. Less than 6% of freshly harvested PMN exhibited characteristics of apoptosis. On aging in culture, day 1 PMN did not undergo apoptosis, whereas 41+/-1 and 29+/-1% of day 2 and 3 PMN, respectively, developed apoptosis, which corresponded to increased ingestion by Mphi. All three Mphi populations engaged different receptor-ligand pairs for the recognition and phagocytosis of PMN. Results indicate the resistance of early wound PMN to age-induced apoptosis, demonstrate wound-Mphi phagocytosis of wound PMN, and identify distinct receptor utilization by wound and other Mphi to ingest wound PMN.

Regulation of arginase isoforms I and II by IL-4 in cultured murine peritoneal macrophages.

Macrophages can express two arginase isoforms with distinct subcellular localization (cytosolic AI and mitochondrial AII). These isoforms are products of different genes and are capable of differential induction. Experiments were performed to identify the specific arginase isoforms induced by interleukin (IL)-4, a Th2 cytokine shown by others to increase arginase activity in macrophages, and serum. Results indicate IL-4, in concert with serum, increases AI, but not AII, mRNA in cultured murine macrophages. Moreover, they show serum to induce both arginase isoforms and to be required for maximal AI induction by IL-4. Together with the enhanced expression of AI, IL-4 induced the expression of the cationic amino acid transporter MCAT-2 and increased L-arginine transport into the cells. Present results confirm, then, specificity in the ability of macrophage arginase isoforms to be induced by different stimuli. Moreover, they suggest that a decrease in intracellular L-arginine concentration resulting from its consumption by arginase may be repaired by concurrent increases in L-arginine influx into the cell.

Recycling cell surface glycoproteins undergo limited oligosaccharide reprocessing in LEC1 mutant Chinese hamster ovary cells.

The ability of particular cell surface glycoproteins to recycle and become exposed to individual Golgi enzymes has been demonstrated. This study was designed to determine whether endocytic trafficking includes significant reentry into the overall oligosaccharide processing pathway. The Lec1 mutant of Chinese hamster ovary (CHO) cells lack N -acetylglucosaminyltransferase I (GlcNAc-TI) activity resulting in surface expression of incompletely processed Man5GlcNAc2 N -linked oligosaccharides. An oligosaccharide tracer was created by exoglycosylation of cell surface glycoproteins with purified porcine GlcNAc-TI and UDP-[3H]GlcNAc. Upon reculturing, all cell surface glycoproteins that acquired [3H]GlcNAc were acted upon by intracellular mannosidase II, the next enzyme in the Golgi processing pathway of complex N -linked oligosaccharides (t1/2= 3-4 h). That all radiolabeled cell surface glycoproteins were included in this endocytic pathway indicates a common intracellular compartment into which endocytosed cell surface glycoproteins return. Significantly, no evidence was found for continued oligosaccharide processing consistent with transit through the latter cisternae of the Golgi apparatus. These data indicate that, although recycling plasma membrane glycoproteins can be reexposed to individual Golgi-derived enzymes, significant reentry into the overall contiguous processing pathway is not evident.

Effect of IL-6 overexpression on the metastatic potential of rat hepatocellular carcinoma cells.

BACKGROUND: Previous studies demonstrated that excess IL-6 production correlated with the metastatic potential of rat hepatocellular carcinoma cells. In the work reported here a retroviral construct containing the gene for murine IL-6 was introduced into otherwise nonmetastatic tumor cells to directly determine the effect of IL-6 overexpression on tumor metastatic potential. METHODS: The clonal cell lines 1682.C.2.9.L0 (L0, poorly metastatic) and 1682.C.2.9.L10 (L10, highly metastatic) were selected from a parental hepatocellular carcinoma induced in ACI rats by feeding an ethionine-containing diet. Viral supernatant was used to infect the PA317 amphotropic cell line, and retrovirus produced from these cells infected the poorly metastatic L0 hepatocellular carcinoma cell line. Neomycin-resistant cells were selected in G418 and designated L0-IL-6. RESULTS: As determined by bioassay, L0 cells produce 10 +/- 1.2 U/mL IL-6 in culture, whereas L10 cells release 95 +/- 11 U/mL (P < 0.01, Student's t-test). Retroviral-mediated IL-6 gene transfer resulted in the production of 1266 +/- 48 U/mL IL-6 by L0-IL-6 cells under identical culture conditions. When an inoculum of 5 x 10(6) cells is injected subcutaneously, both L0 and L10 cell lines result in primary tumors with equivalent rates of growth; only L10 cells metastasize to the lung, however. A similar inoculation of L0-IL-6 cells produced local tumors in all 24 animals tested. Interestingly, 15 of 24 (62%) animals presented with metastatic nodules in the abdominal cavity, whereas no such tumors were found in animals receiving L10 cells. CONCLUSION: Overexpression of IL-6 increases metastatic potential of tumor cells, with preferential metastases to the abdominal cavity when compared with tumor cells elaborating endogenous IL-6.

Wound-induced tumor progression: a probable role in recurrence after tumor resection.

OBJECTIVE: To determine the effect of several wound factors on melanoma growth in a mouse model. DESIGN: Cohort analytic study. SETTING: Animal research facility of Roger Williams Medical Center, Providence, RI. STUDY GROUP: Seventeen groups of 5 C57BL/6 mice each. INTERVENTIONS: A surgical wound was created in 1 hind limb, after which different concentrations of B16F10 melanoma cells were injected in adjacent subcutaneous tissue. The nonwounded hind limb in the same mouse served as a control. In this fashion, a critical tumor cell dose was determined that showed tumor growth in the wounded but not the control hind limb. Tumor growth in control hind limbs then was compared with that in the "artificially wounded" hind limbs, which were co-injected with mouse wound fluid or growth factors. Early (day 1) and late (day 10) wound fluids and tumor growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF), both combined, and interleukin 6 (IL-6) were used. MAIN OUTCOME MEASURE: Wound factors increase tumor growth, indicating potentiation of tumor recurrence at a surgical wound. RESULTS: The critical tumor cell dose was 10(3) cells. All growth factors and both wound fluids showed increased tumor growth over time except IL-6. Hind limbs injected with early wound fluid showed increased tumor growth over time when compared with those injected with late wound fluid (P<.001), TGF-beta (P<.001), bFGF (P<.001), and IL-6 (P<.001). Combined TGF-beta and bFGF co-injection resulted in increased tumor growth compared with TGF-beta (P<.001) and bFGF (P<.001), but did not differ significantly from early wound fluid (P<.07). CONCLUSIONS: The healing wound and its mediators in wound fluid or purified growth factors significantly enhanced tumor growth. Combining TGF-beta and bFGF increased tumor growth to a level closer to wound fluid. The inflammatory response provoked by wound healing mediators may be an important mechanism in tumor growth after ablative surgery.

Distinct arginase isoforms expressed in primary and transformed macrophages: regulation by oxygen tension.

Experiments were performed to identify arginase isoforms expressed in primary and transformed rodent macrophages and to determine the molecular mechanisms for the previously observed increase in arginase activity in macrophages cultured in hypoxia or anoxia. Results demonstrate the following: 1) mRNA and protein for hepatic-type AI arginase are expressed in primary cultures of rat and mouse peritoneal macrophages and are enhanced seven- and nine-fold, respectively, by lipopolysaccharide (LPS). 2) mRNA for extrahepatic-type AII arginase is constitutively expressed in mouse, but not rat, peritoneal macrophages and is detected in RAW264.7 cells after LPS treatment; neither J774A.1 nor P388D1 cells contain arginase mRNA. 3) AI arginase mRNA, arginase activity in cell lysates, and L-arginine flux through arginase in intact cells are all increased in rat wound-derived and mouse peritoneal macrophages by hypoxic or anoxic culture; AII arginase mRNA is, in contrast, suppressed > 50% by O2 deprivation. 4) Expression of the L-arginine transporter mCAT-2 is increased greater than twofold by reduced O2 culture. These results demonstrate substantial variability in arginase isoform expression among primary and transformed rodent macrophages. They also identify AI and AII arginase and the mCAT-2 L-arginine transporter as O2-regulated genes.

Role of nitric oxide in mediation of macrophage cytotoxicity and apoptosis.

Macrophages can recognize and eliminate tumor cells. To this effect, these cells use a variety of cytotoxic effectors. Recent work has paid particular attention to nitric oxide (NO) and its metabolic by-products in mediating macrophage tumor cytotoxicity. Moreover, work from this and other laboratories have indicated that macrophage-dependent, NO mediated tumor cell death meets the morphologic and molecular criteria that define apoptotic cell death. This review will initially discuss the characteristics of macrophage tumor cytotoxicity and the potential mechanisms by which NO can induce apoptosis in tumor cells. In addition, observations of spontaneous and acquired resistance to NO will be analyzed. Lastly, the relevance of results obtained using animal cells to the biology of the human macrophage will be considered.

Effects of lambda-carrageenan induced experimental enterocolitis on splenocyte function and nitric oxide production.

A lambda-carrageenan induced rat model of inflammatory bowel disease (IBD) allowed evaluation of progressive histopathological changes over time and comparison with human disease. In addition, this model was used to test the hypothesis that the immunosuppression observed in IBD may be mediated in part by augmented production of nitric oxide by splenic lymphocytes. Male Sprague-Dawley rats were fed standard rat chow and a drinking solution of 2% lambda-carrageenan without prior sensitization to the compound. At 2, 4, 6, and 8 weeks, small and large intestines were removed for histological examination, and splenocytes were assayed for response to various mitogens and for nitrite production. Intestinal lesions gradually developed in lambda-carrageenan fed rats and were found to be morphologically similar to those observed in human ulcerative colitis. The proliferative response of splenocytes to known mitogens was significantly diminished in carrageenan fed rats, when compared with pair-fed controls, but was fully restored when cultured in the presence of NG-monomethyl-L-arginine, a specific inhibitor of nitric oxide synthase. Preliminary data suggest that overproduction of nitric oxide may provide a molecular mechanism for the immunosuppression observed during chronic inflammatory bowel disease. Further characterization of a reproducible rat model of IBD will allow further investigation into the pathogenesis of the human disease.

NO is not sufficient to explain maximal cytotoxicity of tumoricidal macrophages against an NO-sensitive cell line.

Nitric oxide (NO) is a macrophage cytotoxic effector. Results presented here, however, demonstrate that NO does not fully explain macrophage cytotoxicity against NO-sensitive cells because (1) inhibition of NO production by activated macrophages reduces, not eliminates, cytotoxicity; (2) NO produced chemically in amounts equimolar to those released from macrophages fails to lyse P815 cells; and (3) macrophages isolated from wounds 10 days after injury generate NO just as tumoricidal activated macrophages but are not tumor cytotoxic. The noncytotoxic nature of these wound-derived macrophages is not explained by the release of inactive forms of NO, because they suppress lymphocyte proliferation in an NO-dependent manner, nor by the production of cytoprotective molecules, because their addition to activated macrophage-tumor cell cocultures does not quench cytotoxicity. Interestingly, cytotoxicity can be aroused in day 10 wound-derived macrophages by culture with lipopolysaccharide, and macrophages harvested earlier in the development of the wound are cytotoxic. By generating NO but not killing an NO-sensitive cell, day 10 wound-derived macrophages demonstrate that NO production is not sufficient to account for the killing of an NO-sensitive tumor by macrophages.

B cell lymphoma-2 transfected P815 cells resist reactive nitrogen intermediate-mediated macrophage-dependent cytotoxicity.

Activated murine peritoneal macrophage cytotoxicity against P815 tumor cells has been shown to be mediated by the reactive nitrogen intermediates (RNI) produced by macrophages from L-arginine through nitric oxide (NO) synthase. Previous results from this laboratory indicated that NO-dependent killing of P815 fulfilled the criteria for apoptotic death. Work by others, in turn, demonstrated that the product of the bcl-2 gene confers protection against various inducers of apoptosis, including reactive oxygen intermediates. Experiments were performed to determine whether Bcl-2 could equally protect sensitive cells from RNI-dependent apoptosis within the context of a relevant biologic system such as the delivery of such RNI by activated macrophages. Results demonstrated that transfection of P815 cells with the human bcl-2 gene confers immunity from RNI-dependent, macrophage-mediated cytotoxicity. In contrast with wild-type or mock-transfected P815 cells, which do not contain detectable Bcl-2, bcl-2-transfected cells showed minimal DNA fragmentation and cell membrane failure when cocultured with activated macrophages. Additional findings indicate that Bcl-2 affords the transfected cells almost complete resistance to the DNA-fragmenting effects of chemically generated NO or H202 and partial protection from their cytolytic effects. These findings are consistent with the hypothesis that tumor cells expressing bcl-2 may escape destruction by macrophage-dependent immune surveillance mechanisms.

Interleukin-6 production by rat hepatocellular carcinoma cells is associated with metastatic potential but not with tumorigenicity.

BACKGROUND: The phenotypic characteristics that allow some tumor cells to metastasize have not been fully identified. The production and/or response of tumor cells to various growth factors have been shown to distinguish cells of differing metastatic potentials. OBJECTIVES: To determine (1) whether rat hepatocellular carcinoma cell lines produce interleukin-6 (IL-6) and (2) whether production of IL-6 correlates with either metastatic potential or tumorigenicity. METHODS: The clonal cell lines 1682.C.2.9.L0 (poorly metastatic) and 1682.C.2.9.L10 (highly metastatic) were selected from a parental hepatocellular carcinoma induced in ACI rats by feeding an ethionine-containing diet and adapted to growth in vitro. RESULTS: Both cell lines resulted in primary tumors with equal frequency and developed a 40-mm nodule in a similar period time, when an inoculum of 5 X 10(6) cells was injected subcutaneously; however, only L10 cells metastasized to the lung. These cell lines did not demonstrate differential expression of several antigens noted to correlate with metastatic potential, including CD44 variant glycoprotein, p53, transferrin receptor, and E-cadherin. In contrast, L0 cells produced less than 10 U of IL-6 per milliliter in culture (as determined by bioassay using 7TD1 cells), whereas L10 cells released more than 95 U of this cytokine per milliliter under identical culture conditions (P<.01, Student's t test). In addition, serum concentrations of IL-6 were elevated in animals bearing L10-induced primary tumors but not in those with L0-induced tumors of comparable mass. Exogenous addition of IL-6 to both tumor cell lines had no effect on the rate of growth in vitro, supporting the similar the tumorigenic potentials observed in vivo. CONCLUSION: Excess IL-6 production appears to identify cells with metastatic potential and does not appear to be essential to the establishment of a primary tumor.

Electron transport chain activity in normal and activated rat macrophages.

The pivotal role played by the macrophage in specific and nonspecific immunity suggests that the physiological status of the macrophage may effect the overall regulation of the host defense system. Many studies have evaluated macrophages as effector cells by examining expression of surface markers, cytokine release, or tumor killing in the presence of challenge to host defenses. In this report, the physiological parameter of mitochondrial respiration in freshly isolated rat macrophages is shown to be regulated upon activation in vivo. Assay conditions for the reduction of MTT [3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide] in rat macrophages were optimized and used to quantitate electron transport chain activity as a measure of mitochondrial respiration. Corynebacterium parvum administration significantly increased the activity of the mitochondrial electron transport chain in both peritoneal (120% increase, 0.18 +/- .01 vs 0.40 +/- .03, P < 0.01) and liver macrophages (143% increase, 0.12 +/- .02 vs 0.30 +/- .06, P < 0.01) as detected by augmented MTT reduction. It is demonstrated further that MTT reduction is distinct from the respiratory burst activity of macrophages and supports the mitochondrial localization of intracellular MTT reduction in this cell type. These results demonstrate that electron transport chain activity is a physiological indicator of macrophage activation.