Distinct inflammatory responses of adherent vascular lung neutrophils to pulmonary irritants.

The nature and the extent of the damage that occurs in the lung following exposure to pulmonary irritants vary with the pathogenic agent. In the present studies we determined if this was due to unique functional responses of adherent vascular neutrophils to different irritants. Because of their location within the lung, these cells may be more relevant than circulating neutrophils to the pathophysiology of irritant-induced lung injury. For our studies we used two model irritants, ozone and endotoxin, which cause distinct pathologic effects in the lung. Treatment of rats with ozone resulted in a transient increase (2-fold) in the number of adherent vascular neutrophils in the lung which was maximum 2 hr after exposure and returned to control levels by 12 hr. In contrast, following endotoxin administration, 10-fold greater numbers of adherent neutrophils were recovered from the lung. Moreover, cell number remained elevated 3-fold for up to 48 hr. Unstimulated neutrophils isolated 2-12 hr after endotoxin treatment of rats produced 3 times more superoxide anion than cells from ozone-treated rats. Cells isolated 12-48 hr after endotoxin administration were also sensitized to produce more nitric oxide than cells from ozone-treated rats and to express inducible nitric oxide synthase protein. These data demonstrate that endotoxin and ozone induce distinct patterns of accumulation and functional changes in adherent vascular neutrophils in the lung which may contribute to different pathological processes observed following exposure to these pulmonary irritants.

Macrophage and interleukin-1 induced nitric oxide production and cytostasis in hamster tumor cells varying in malignant potential.

Nitric oxide has been shown to contribute to cytotoxicity in mouse and rat tumor cells. In these studies we examined the role of nitric oxide in cytostasis in hamster tumor cells varying in their malignant potential. Spontaneously transformed hamster embryonic fibroblasts (STHE cells) with low metastatic activity produced significantly greater amounts of nitric oxide in response to interleukin-1 (IL-1) or lipopolysaccharide (LPS)-activated hamster alveolar macrophages (HAM) than did tumor cell lines with high experimental metastatic activity (HET-SR, HET-SR1, STHE-83/20 cells). HET-SR cells, which exhibit low spontaneous metastastic activity, also produced relatively high levels of nitric oxide in response to IL-1, whereas the response of the spontaneously metastatic lines, HET-SR1 and STHE-83/20 cells, was low. IL-1 and HAM also induced cytostasis in nitric oxide-producing STHE and HET-SR cells. However, the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), had no effect on this activity. These findings, together with the observation that anti-tumor necrosis factor alpha antibody prevented HAM-mediated cytostasis in all of the tumor cell lines demonstrate that nitric oxide is not involved in hamster macrophage-induced tumor cell growth suppression. In contrast to HAM, rat alveolar macrophages, which produced nitric oxide in response to LPS, exerted similar levels of cytostasis toward all of the hamster tumor cell variants, an action that was blocked by L-NMMA in HET-SR, HET-SR1, and STHE-83/20 cells. Thus production of nitric oxide by hamster tumor cells is inversely correlated with their malignant potential. However, nitric oxide does not appear to be involved in IL-1- or HAM-mediated cytostasis toward hamster tumor cells.

Cytostasis is required for IL-1 induced nitric oxide production in transformed hamster fibroblasts.

Interleukin-1 (IL-1) is known to inhibit proliferation in some tumor cells. This proinflammatory cytokine also induces nitric oxide production in a variety of cell types. In the present studies we determined if nitric oxide is involved in IL-1 induced growth inhibition in spontaneously transformed hamster embryonic fibroblasts (STHE cells). Both IL-1 alpha and IL-1 beta were found to stimulate nitric oxide production and to reduce 3H-thymidine (TdR) incorporation in high density cultures of STHE cells. However, maximal cytostasis was observed at least 24 h before significant amounts of nitric oxide accumulated in the cultures. In addition, doses of IL-1 which were too low to stimulate nitric oxide synthesis were effective in inducing cytostasis. Furthermore, in low density cultures of STHE cells, IL-1 inhibited DNA synthesis without inducing nitric oxide production. The nitric oxide synthase inhibitor NG-monomethyl-1-arginine (L-NMMA) had no effect on proliferation of cells plated at low density. In contrast, L-NMMA treatment resulted in a 40-60% reduction in IL-1 induced cytostasis in high density cultures. Neutralizing antibodies to IL-1 were found to completely block IL-1 induced cytostasis and nitric oxide production in cells plated at both densities. Although anti-IL-1 alpha and anti-IL-1 beta antibodies were highly specific and did not cross react, anti-tumor necrosis factor-alpha (TNF-alpha) antibody was able to partially suppress activation of STHE cells by both IL-1 alpha and IL-1 beta. These data suggest a potential involvement of endogenous TNF-alpha in IL-1 induced cytostasis and nitric oxide production. Exponentially growing STHE cells produced six-times less nitric oxide than non-proliferating cells. A ten-fold excess of 1-arginine was found to stimulate nitric oxide synthesis, an action that was independent of the rate of cellular proliferation. Taken together these data suggest that nitric oxide is not a major mediator of IL-1 induced cytostasis in STHE cells. Moreover, cytostasis appears to be required for nitric oxide synthesis in these cells.

Mechanisms regulating macrophage-induced nitric oxide production by spontaneously transformed hamster fibroblasts.

Nitric oxide has been implicated as an important effector molecule involved in tumor cell growth and cytotoxicity. In these studies we examined mechanisms regulating nitric oxide production by hamster tumor cells. Cocultures of hamster alveolar macrophages (HAM) and spontaneously transformed hamster embryonic fibroblasts (STHE cells) produced significant quantities of nitric oxide in response to lipopolysaccharide (LPS). Culture supernatants from HAM treated with LPS also stimulated nitric oxide production by STHE cells, whereas tumor cell culture supernatants had no effect on HAM. These data, together with the findings that paraformaldehyde treatment of STHE cells, but not macrophages, completely abrogated nitric oxide production in the cocultures demonstrate that the tumor cells were the source of this mediator. In contrast to STHE cells, STHE-83/20 cells, a highly malignant variant, did not produce nitric oxide in response to HAM or HAM culture supernatants even in the presence of LPS. Both anti-tumor necrosis factor-alpha (TNF-alpha) and anti-interleukin-1alpha (IL-1alpha) antibodies inhibited HAM-induced nitric oxide production by STHE cells. However, the kinetics of their effects were different. Moreover, although the nitric oxide stimulating activity in HAM culture supernatants was abrogated by anti-TNF-alpha antibody, it was only minimally reduced by anti-IL-1alpha antibody. These data demonstrate that TNF-alpha and IL-1alpha play distinct roles in induction of nitric oxide synthesis in STHE cells. HAM were also found to suppress proliferation of STHE cells, an effect that was inhibited by anti-TNF-alpha antibody, but not NG-monomethyl-L-arginine, which blocks nitric oxide synthase. Abrogation of macrophage-induced cytostasis in STHE cells by anti-TNF-alpha antibody was associated with decreased nitric oxide production. Thus TNF-alpha released by macrophages may indirectly activate STHE cells for nitric oxide synthesis by suppressing tumor cell proliferation.

Unique patterns of regulation of nitric oxide production in fibroblasts.

The pathways regulating rat and mouse embryonic and lung fibroblast nitric oxide production were analyzed in an attempt to evaluate the potential role of these cells in nonspecific host defense and inflammation. Interleukin-1 beta (IL-1 beta) was found to be the strongest single activator in all types of fibroblasts examined. In addition, lipopolysaccharide (LPS) was synergistic with IL-1 beta or tumor necrosis factor-alpha (TNF-alpha) in induction of nitric oxide synthesis. These patterns of responsiveness are not observed in macrophages and may be significant in initiation of early host defense processes, before specific interferon-gamma (IFN-gamma)-mediated immune responses have become operative. Rat and mouse fibroblasts were also found to produce nitric oxide when primed with IFN-gamma and simultaneously treated with IL-1, TNF-alpha, or LPS. The doses of IFN-gamma effective in priming fibroblasts for nitric oxide production were as low as 1-10 U/ml. Furthermore, effective triggering doses of LPS, TNF-alpha, and IL-1 were 10 ng/ml, 100 U/ml, and 0.2 ng/ml, respectively. These results demonstrate that fibroblasts are activated more readily to produce nitric oxide than interstitial macrophages and may be the major source of this mediator in tissues. Immunohistochemical studies demonstrated that fibroblasts are heterogeneous with respect to inducible nitric oxide synthase expression with the majority of cells not involved in the response. Fibroblasts were also found to be distinct from macrophages in their sensitivity to the suppressive effects of transforming growth factor-beta, which in fibroblasts inhibited both IFN-gamma plus LPS- and IFN-gamma plus TNF-alpha-induced nitric oxide production. At the stage of growth crisis, a dramatic increase in nitric oxide production was observed in rat fibroblasts in response to IFN-gamma or TNF-alpha that may be directly correlated with cellular senescence. Taken together, our data suggest that mouse and rat fibroblasts are potential effectors in both IFN-gamma-dependent and -independent nitric oxide-mediated processes and that the patterns regulating nitric oxide metabolism in these cells are distinct from those of macrophages.

Functional characterization of interstitial macrophages and subpopulations of alveolar macrophages from rat lung.

The specific function of interstitial macrophages (IM) in the lung is poorly understood because of difficulties in isolating these cells in high purity or large number. In the present studies, a pure population of enzymatically isolated IM and lung macrophages obtained mechanically from the lung were compared functionally with alveolar macrophages recovered by lavage (AM). Macrophages isolated mechanically from the tissue and AM displayed similarly high levels of Fc-receptor mediated phagocytosis. In contrast, IM phagocytized significantly fewer opsonized sheep red blood cells per macrophage than AM. In addition, although some variations in the amounts of nitric oxide and superoxide anion produced by AM and macrophages obtained by mechanical tissue disruption were observed, these subpopulations released significantly more of these mediators than IM. These data support the concept that macrophages isolated by mechanical disruption of the tissue represent a subpopulation of AM. We also found that, in contrast to AM, IM did not respond synergistically to combinations of IFN-gamma and lipopolysaccharide (LPS) or tumor necrosis factor alpha in terms of nitric oxide production. Furthermore, regulation of superoxide anion release in AM and IM by LPS and/or IFN-gamma was distinct. Taken together, these studies demonstrate that IM are functionally different from other macrophage subpopulations which might reflect their unique location within the lung.

A single exogenous stimulus activates resident rat macrophages for nitric oxide production and tumor cytotoxicity.

Based on experiments with mouse cells, it appears that macrophage activation for cytotoxicity requires two exogenous signals. One signal primes or sensitizes the cells, while the second activates them for killing. The present studies were designed to analyze the capacity of rat macrophages to be activated for nitric oxide production and for cytotoxicity by different inflammatory stimuli. We found that both resident alveolar macrophages (AMs) and resident peritoneal macrophages (PMs) from Sprague-Dawley rats produced nitric oxide in response to relatively low doses of a single exogenous activating stimulus [interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS)]. Resident PMs treated with either of these agents alone also exhibited nitric oxide-mediated cytotoxicity toward xenogeneic and allogeneic tumor targets. In contrast to results reported previously with both resident and elicited PMs from mice, tumor necrosis factor-alpha (TNF-alpha) exerted only a small enhancing effect on IFN-gamma-induced nitric oxide production by resident rat PMs. In addition, although some level of cooperativity between IFN-gamma and LPS was observed at low concentrations of LPS (< 10 ng/ml), IFN-gamma did not augment the effects of higher concentrations of LPS (> or = 10 ng/ml) on nitric oxide production by PMs. In contrast to PMs, AMs had a strong synergistic response to combinations of IFN-gamma and LPS or TNF-alpha but also only at relatively low concentrations of IFN-gamma and LPS. Furthermore, maximum nitric oxide production induced by IFN-gamma in these cells could be enhanced by TNF-alpha or low doses of LPS. However, as observed with PMs, combinations of IFN-gamma and higher doses of LPS did not significantly augment maximum nitric oxide production induced in AMs by LPS alone. Thus, our data suggest that resident rat PMs and AMs resemble elicited mouse PMs in their ability to respond to a single activating stimulus. However, PMs and in some respects AMs differ from the latter by their reduced responsiveness to combinations of activators. Taken together, our results suggest that two exogenous stimuli are not required for full activation of resident macrophages from Sprague-Dawley rats.

Isolation and partial characterization of subpopulations of alveolar macrophages, granulocytes, and highly enriched interstitial macrophages from rat lung.

The contribution of interstitial macrophages (IM) to lung defense, homeostasis, and pathophysiology is for the most part unknown. Studies on this cell type are difficult because they are not readily accessible in large numbers or in high purity. In the present work, various nonenzymatic and enzymatic methods were compared with the aim of isolating and characterizing pure populations of lung IM. The results of our studies demonstrate that most procedures currently used to isolate IM yield subpopulations of alveolar macrophages (AM) or IM highly contaminated by AM and granulocytes. We found that lavage of the lung yielded only one half of the total AM present in the tissue. The remainder of the AM could only be obtained by extensive washing of cut and disaggregated lung tissue, which is considered by some investigators to be an effective procedure for IM isolation. According to our results, cells recovered by lavage and washing of cut and disaggregated lung tissue were morphologically and histochemically identical, were strongly positive for nonspecific esterase, highly phagocytic, and appeared to represent subpopulations of AM with apparently varying degrees of adherence to the alveolar walls. We also found that IM could be obtained in high purity by sequential digestion of the remaining lung tissue with 60 and 175 IU/ml of collagenase followed by selective adherence. Digestion of the tissue with 60 IU/ml of collagenase resulted in a highly enriched population of granulocytes and also reduced their contamination in the IM population. The resulting IM were distinct from AM by morphology and histochemistry. Like AM, these cells displayed Fc receptor-mediated phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

[Production of antitumor cytostatic factors by nonactivated resident macrophages of the peritoneal cavity of Syrian hamsters]. / Produktsiia protivoopukholevykh tsitostaticheskikh faktorov neaktivirovannymi rezidentnymi makrofagami peritoneal'noi polosti siriiskikh khomiakov.

The production of soluble cytostatic (CS) factor(s) by nonactivated peritoneal resident macrophages (Mph) of Syrian hamsters was found with the use of susceptible spontaneously transformed in vitro cells of STHE cell strain. The CS factor was determined by two modifications of CS test: 1) incorporation of 3H-TdR to the nuclei of target cells and 2) direct determination of the number of cells in the wells. The selected in vivo highly malignant variant of STHE strain appeared to be resistant to CS factors Mph.

[Susceptibility of cells with different stage of malignancy to cytostatic action of resident peritoneal macrophages of Syrian hamsters]. / Chuvstvitel'nost' kletok raznoi stepeni zlokachestvennosti k tsitostaticheskomu deistviiu rezidentnykh peritoneal'nykh makrofagov siriiskikh khomiakov.

Syrian hamster non-activated resident peritoneal cells (PC) and peritoneal macrophages (Mph) was demonstrated. The in vivo selection of highly tumourigenic and highly metastatic variants of this strain correlated with their resistance to CSA PC and Mph in four cell variants out of five examined. The highly tumourigenic Syrian hamster embryo cells in vitro transformed by Rous sarcoma virus were highly resistant to CSA PC without selection in vivo. The resistance of highly malignant cells to CSA PC appeared to be unrelated to their ability to produce immunosuppressing prostaglandins of E type.

[Cytostatic action of the peritoneal cells of Syrian hamsters on transformed cells]. / Tsitostaticheskoe deistvie peritoneal'nykh kletok siriiskikh khomiakov na transformirovannye kletki.

The cytostatic effect (CSE) of intact Syrian hamster peritoneal cells (PC) was determined by their capability to inhibit 3H-thymidine incorporation in target cells of HETR, which were placed in 1 X 10(4) or 4 X 10(4) cells per well together with 4 tenfold differing concentrations of PC (10(2)-10(5]. The optimum of CSE was seen with the use of maximal doses of PC and HETR in reaction. Maximal level of CSE with all effector-target cell rations was observed between 23-28 hours of contact. These data permit to suggest the role of HETR cells in activation of PC, as well as the transfer of cytostatic state in dense cell shift mediated by cell-cell contacts. The role of humoral cytostatic factor is also not excluded.

[Cytostatic action of the peritoneal cells of Syrian hamsters to transformed and tumor cells in vitro and its suppression by prostaglandin E2]. / Tsitostaticheskoe deistvie peritoneal'nykh kletok siriiskikh khomiakov na transformirovannye i opukholevye kletki in vitro i ego podavlenie prostaglandinom E2.

High antitumour activity of Syrian hamster peritoneal cells (PC) is demonstrated in the cytostatic test in vitro. The cytostatic effect (CSE) of PC on hamster cell strains can be revealed at the very early stage (7 hours of cocultivation) and it remains stable during at least 24 hours. Single intraperitoneal injection of physiological (10(-9) M) or pharmacological (10(-7)) doses of prostaglandin E2 preparation leads to the prolonged and significant inhibition of CSE of PC.

[Migration activity of peritoneal exudate cells of Syrian hamsters at different times after suppression of their natural resistance to tumors]. / Migratsionnaia aktivnost' kletok peritoneal'nogo ékssudata siriiskikh khomiakov v razlichnye sroki posle podavleniia ikh estestvennoi rezistentnosti k opukholi.

A study was made of migration activity (MA) of peritoneal macrophages of Syrian hamsters after depression of their antitumor natural resistance (NR) induced by injection by heat-inactivated tumor cells. The MA and depression of NR were most pronounced between day 14 and day 20 after inoculation of the animals with inactivated tumor cells of E-1 and STHE-LM8 tumor cells. Inoculation of the hamsters with heat-inactivated tumor cells of another strain (parenteral STHE) did not induce NR depression or enhanced MA of peritoneal macrophages of the treated animals. It is concluded that depression of antitumor NR essential for tumor induction and growth is apparently connected with alterations in the functional activity of macrophages, possibly with their suppressor activity.

[Migration activity of peritoneal exudate cells in Syrian hamsters in normal conditions and during suppression of their natural resistance to tumors]. / Migratsionnaia aktivnost' kletok peritoneal'nogo ékssudata siriiskikh khomiakov v norme i pri podavlenii ikh estestvennoi rezistentnosti k opukholi.

Migration activity (MA) of peritoneal exudate cells (PEC) was studied in Syrian hamsters in normalcy and under intraperitoneal injection into the animals of inactivated normal and tumor cells including those capable of inhibiting the natural animals' resistance to tumor. No significant individual differences were found in MA of PEC of intact hamsters. MA of PEC of hamsters treated with inactivated normal or spontaneously transformed in vitro cells of hamster embryo did not differ from the means of MA in the control. MA of PEC of hamsters treated with inactivated tumor cells was found to be appreciably enhanced. The ability of inactivated tumor cells to induce the enhancement of MA of PEC correlates with their ability to suppress natural tumor resistance.

[Membrane antigens of transformed and tumor cells of hamsters]. / Izuchenie membrannykh antigenov transformirovannykh i opukholevykh kletok khomiaka.

Normal membrane antigens to tumour and in vitro transformed tissue cultures of Syrian hamster cells were comparatively studied in cross-adsorption experiments. It was found that each cell strain has an individual profile of normal membrane antigens with characteristic quantitative and qualitative features seen on antigen expression. The absence of at least one of the antigens which the cells of other strains possessed was demonstrated with reference to the cells of each line tested. All 5 tumor cell lines were bearing antigen or a group of antigens absent on normal embryonic hamster cells (HE) and 5-strains of in vitro transformed HE cells.