In vivo neutrophil emigration in response to interleukin-1 and tumor necrosis factor-alpha

The migration of polymorphonuclear leukocytes (PMN) in response to recombinant interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF), C5a, and f-met-leu-phe-lys (FMLPL) in vivo was studied using a mouse subcutaneous sponge implantation model. In this model sponges were implanted in C3H/OUJ mice, and 2 days later they were injected with the test sample. After varying times, sponges were removed and digested with collagenase, and total cell counts and differentials were enumerated. IL-1 was found to stimulate a significant influx of PMN, which peaked at 6 hr and declined to near baseline levels by 24 hr. This response was dose-dependent, with the greatest response observed when 5 units of IL-1 were injected. When the IL-1 concentration was increased to 10 U, the total number of PMN migrating into the sponge was decreased, compared with that observed with 5 U of IL-1. The overall number of PMN migrating into the sponge 6 hr after injecting 5 U of IL-1 averaged 269% of the number of PMN migrating randomly into the sponge. No difference in the total number of macrophages or lymphocytes in control or IL-1-injected sponges was observed in this time frame. Heat treatment of the IL-1 at 90 degrees C for 30 min ablated the response. Similar studies with TNF and C5a showed that both of these agents also stimulated an influx of PMN that peaked 6 hr postinjection. In contrast, FMLPL did not stimulate a PMN response. When IL-1 and TNF were injected simultaneously, an additive response was observed. These data indicate that IL-1, TNF, and C5a can all stimulate a PMN response in vivo and support the hypothesis that these substances are actively involved in the mobilization of PMN to inflammatory sites in vivo.     

Increased expression of tumor necrosis factor-alpha in diabetic macrovasculopathy.

Large vessel disease, a common feature of diabetes mellitus, appears to run an aggressive course, but its cellular and molecular aspects remain partially elucidated. Although in common atherosclerosis and especially in other forms of accelerated vasculopathy, immunoinflammatory mechanisms participate in the disease process, it is unclear whether this is present in diabetic vasculopathy. We hypothesized that diabetic macrovasculopathy, compared with classical atherosclerosis, is associated with increased immunoinflammatory features and matrix accumulation. In this study, vessel segments obtained after lower-limb amputation for advanced atherosclerotic disease, from type 2 diabetic patients (n = 20; 68.9+/-10.9 years) and nondiabetic patients (n = 16; 67.1+/-14.6 years) were analyzed. Histological characteristics (extent of intimal proliferation, cellularity, and fibrosis) were semiquantitatively graded in the two lesion types. Using immunohistochemistry, the presence of T cells and macrophages, accumulation of fibronectin, and expression of tumor necrosis factor-alpha was also assessed. Histological features of these advanced atherosclerotic lesions were similar in the two lesions examined. By immunohistochemistry, a similar pattern of T-cell and macrophage infiltration and fibronectin accumulation was observed. Nevertheless, increased expression of tumor necrosis factor-alpha was observed in diabetic lesions (13/19 patients had positive staining), whereas only 2 of 16 lesions from nondiabetic patients had positive staining (p < 0.003), with an odds ratio of 15.17 (confidence interval 2.12-139.5). These data suggest that increased expression of tumor necrosis factor-alpha observed in the diabetic lesions may reflect an enhanced inflammatory activity associated with the development of vascular lesions in type 2 diabetic patients.     

In vivo preconditioning with normobaric hyperoxia induces ischemic tolerance partly by triggering tumor necrosis factor-alpha converting enzyme/tumor necrosis factor-alpha/nuclear factor-kappaB

Recent studies suggest that intermittent and prolonged normobaric hyperoxia (HO) results in brain ischemic tolerance (BIT), reducing ischemic brain injury. We have attempted to determine the time course of HO-induced BIT, and to explore the putative roles of tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE), TNF-alpha, and nuclear factor-kappaB (NF-kappaB) activation in mediating this effect. Two core experimental protocols were applied to rats (experiments 1 [E1] and 2 [E2] respectively). E1 rodents comprised six subgroups, breathing room air (RA; O(2)=21%), or 95% oxygen (HO) for 4, 8, 16 h (4RA, 8RA, 16RA and 4HO, 8HO, 16HO respectively). E2 rodents were divided into subgroups, exposed to 95% inspired HO for 4 h/day for six consecutive days (intermittent hyperoxia, InHO) or for 24 continuous hours (prolonged hyperoxia, PrHO). Each of these had a control group exposed to 21% oxygen in the same chamber. Twenty-four hours after pretreatment, each group was randomly divided to receive 60 min right middle cerebral artery occlusion (MCAO-operated), sham-operation (without MCAO), or no operation (intact). After 24 h reperfusion, neurologic deficit score (NDS), brain water content, Evans Blue extravasation (as a marker of blood-brain barrier permeability), TACE expression, serum TNF-alpha, and phosphor- kappaBalpha levels were assessed in all animals, and infarct volume in the MCAO-operated subgroups. E1: Compared with the control (RA) group, infarct volume was reduced by 58.6% and 64.4% in 16 h and 24 h respectively. NDS and Evans Blue extravasation was also reduced in 16 h and 24 h. There was no statistical difference among 4 h and 8 h. E2: Preconditioning with prolonged and intermittent HO decreased NDS, infarct volume and upregulated TACE and increased phosphor-kappaBalpha and serum TNF-alpha level significantly. Although further studies are needed to clarify the mechanisms of brain ischemic tolerance, InHO and PrHO may partly exert their effects via triggering TACE/TNF-alpha/NF-kappaB.     

Endotoxin-induced cytokine gene expression in vivo. II. Regulation of tumor necrosis factor and interleukin-1 alpha/beta expression and suppression.

Tumor necrosis factor alpha (TNF alpha) mRNA is present in a preformed intracellular pool in the spleen, liver, and small bowel of naive rats. Endotoxin (Salmonella typhus lipopolysaccharide) injected intravenously induces little or no increase in whole-organ TNF mRNA levels at 15', 30', 1 degree, 2 degrees, or 4 degrees, whereas serum TNF levels are markedly elevated at 1 and 2 hours. Dexamethasone pretreatment of rats suppresses LPS-induced serum TNF concentrations, but does not suppress TNF mRNA levels in the spleen or bowel. Tachyphylaxis experiments demonstrate that a second injection of endotoxin 2 hours after an initial injection fails to induce a second peak of serum TNF, although TNF mRNA levels in the spleen and bowel remain at the levels found in naive rats. Corynebacterium parvum upregulates endotoxin-induced serum TNF release and intravenous injection of IL-1 induces the release of serum TNF but neither alters whole-organ TNF mRNA levels. Interleukin-1 alpha (IL-1 alpha) mRNA was not constitutively detected in whole-organ RNA preparations of the spleen, liver, and small bowel of naive rats. Endotoxin induces IL-1 alpha mRNA most easily appreciated in the spleen beginning at 1 hour, peaking at 2 to 4 hours, and disappearing by 6 hours. Interleukin-1 beta (IL-1 beta) mRNA was not constitutively detected in the organs examined or was present in small amounts. Endotoxin induces IL-1 beta mRNA beginning at 0.5 hours, peaking at 1 hour, and disappearing by 6 hours. Dexamethasone pretreatment prevents the LPS-induced appearance of IL-1 alpha mRNA and suppresses but does not completely inhibit the appearance of IL-1 beta mRNA. C. parvum upregulates endotoxin-induced IL-1 mRNA expression. Intravenous injection of TNF or IL-1 both induce IL-1 mRNA expression. In conclusion, TNF mRNA is constitutively expressed and TNF mRNA levels as analyzed in whole-organ RNA preparations do not change in concert with serum TNF protein levels during conditions of endotoxemia, dexamethasone treatment, tachyphylaxis, priming with C. parvum, or after injection of IL-1. In contrast, IL-1 mRNA expression during endotoxemia, dexamethasone treatment, priming with C. parvum, or after injection of TNF or IL-1 shows clear increases and decreases in whole-organ RNA preparations.     

Induction of plasminogen activator inhibitor 1 gene expression in murine liver by lipopolysaccharide. Cellular localization and role of endogenous tumor necrosis factor-alpha.

We previously demonstrated that lipopolysaccharide (LPS) induces plasminogen activator inhibitor 1 (PAI-1) gene expression primarily in endothelial cells in most organs of the mouse, with maximal induction by 3 hours. Here we show that induction in the liver occurs in a distinctly different pattern. For example, the increase in PAI-1 mRNA in liver was biphasic with an initial peak at 1 to 2 hours and a second peak at 6 to 8 hours. Moreover, in situ hybridization experiments revealed that PAI-1 mRNA was induced in both endothelial cells and hepatocytes. The endothelial cell response was monophasic and maximal between 1 and 4 hours, whereas the hepatocyte response was biphasic, peaking at 2 hours and again at 6 to 8 hours. To determine possible mechanisms involved in the induction of PAI-1 by LPS, we analyzed the tissues for changes in tumor necrosis factor (TNF)-alpha LPS caused a rapid induction of TNF-alpha mRNA in Kupffer cells, detectable within 15 minutes. Pretreatment of mice with anti-TNF antiserum before challenge with LPS reduced the subsequent increase in plasma levels of PAI-1 by 50 to 70% and significantly reduced the level of induction of PAI-1 mRNA in the liver at both early and late times. Pretreatment appeared to inhibit induction primarily within hepatocytes. These results suggest that LPS may induce PAI-1 in endothelial cells and hepatocytes by different mechanisms.     

Tumor necrosis factor-alpha antagonism by the murine tumor necrosis factor-alpha receptor 2-Fc fusion protein exacerbates histoplasmosis in mice.

Treatment of some inflammatory conditions with tumor necrosis factor-alpha (TNF-alpha) antagonists is efficacious, but such treatments are associated with infections with intracellular pathogens, including Histoplasma capsulatum. We explored protective immunity to H. capsulatum in mice given a fusion protein consisting of TNF-alpha receptor 2 (TNFR2) bound to the Fc portion of mouse IgG1. Intraperitoneal administration of this inhibitor exacerbated primary or secondary pulmonary infection at dosages ranging from 1 to 5 mg/kg. All mice with primary infection given the inhibitor succumbed to infection within 10-21 days of treatment. In secondary histoplasmosis, mice receiving 1, but not 5, mg/kg survived treatment. Fungal burden was increased even if treatment with the inhibitor was initiated after the onset of infection. The inflammatory response of the lungs of mice given the inhibitor did not differ from that of mice given control vehicle. Susceptibility was not associated with major alterations in cytokines known to protect or exacerbate infection. However, expression of nitric oxide synthase 2 (NOS2) was depressed early in primary infection. These results demonstrate that antagonism of endogenous TNF-alpha by this fusion protein modulates susceptibility. Impaired immunity is not a result of altered cytokine responses or changes in the inflammation and may not be demonstrable in other murine strains.     

Interleukin-4 differentially regulates tumor necrosis factor-alpha gene expression by human T lymphocytes and monocytes.

Tumor necrosis factor-alpha (TNF-alpha), a product of both mononuclear phagocytes and T lymphocytes, is an important proximal mediator of a number of acute and chronic inflammatory disease states. In this investigation we examine the regulatory effects of the lymphocyte product interleukin-4 (IL-4) on the gene expression of TNF-alpha from stimulated human peripheral blood monocytes (PBM) and T lymphocytes. We demonstrated the dose-dependent suppression of TNF-alpha mRNA and protein synthesis from lipopolysaccharide-treated PBM by IL-4. The suppressive effects of IL-4 appear to be dependent upon de novo protein synthesis, as cycloheximide abrogated the IL-4-induced reduction in TNF-alpha mRNA levels from PBM. In contrast to the suppressive effects of IL-4 on PBM-derived cytokine expression, IL-4 did not alter TNF-alpha mRNA expression from alpha-Cd3 or PMA + alpha-CD-28-treated T lymphocytes. Moreover, IL-2 mRNA expression from similarly treated T lymphocytes was unaltered by IL-4. Our findings demonstrate that disparity exists in the regulation of TNF-alpha gene expression from different immune cell populations which may have important implications in the evolution of acute and chronic inflammatory responses.     

Phagocytosis enhances murine macrophage activation by interferon-gamma and tumor necrosis factor-alpha

Previously, we reported that exposure of bone marrow-derived macrophages (M phi) to a phagocytic stimulus in the simultaneous presence of interferon-gamma (IFN-gamma) induced these cells to generate nitrite (NO2-). This effect was achieved using both living (i.e. promastigotes of the protozoan parasite Leishmania enriettii) and inert (latex beads) particles. When the phagocytic stimulus was Leishmania, enhanced intracellular killing accompanied elevated NO2- secretion. As shown here, the capacity of phagocytosis to elicit NO2- production by IFN-gamma-treated M phi was inhibited by antibody to murine recombinant tumor necrosis factor-alpha (rTNF-alpha), suggesting that phagocytosis enabled IFN-gamma to activate M phi via the induction of TNF-alpha as an autocrine second signal. M phi NO2- production in response to rIFN-gamma and either exogenous TNF-alpha or Leishmania was strongly enhanced by prostaglandin E2, consistent with such a mechanism. However, addition of either Leishmania promastigotes or latex beads to M phi cultures simultaneously exposed to both IFN-gamma and exogenous murine or human rTNF-alpha further potentiated activation as measured by NO2- release. Furthermore, anti-TNF antibody failed to inhibit M phi responses to rIFN-gamma and bacterial lipopolysaccharide (LPS) in the presence or absence of Leishmania; also exogenous rTNF-alpha did not significantly affect NO2- production by IFN-gamma/LPS cultures despite a strong enhancement by Leishmania. These results suggest that phagocytosis enhances M phi responses by a process more complex than the sole induction of TNF-alpha. Phagocytosis also increased M phi NO2- production elicited by IFN-gamma plus TNF-alpha in L-arginine-deficient media. These results indicate that phagocytosis may be an important mechanism of up-regulating M phi microbicidal activity, and could be particularly relevant upon arginine depletion which occurs during an inflammatory response.     

Quercetin inhibits LPS-induced nitric oxide and tumor necrosis factor-alpha production in murine macrophages.

High amounts of nitric oxide and TNF-alpha generated by activated macrophages induce several pathophysiological conditions during acute and chronic inflammation. Identification of new pharmacological reagents that can prevent TNF-alpha and/or NO overproduction is of considerable medical interest. In this report we provide evidence that the overproduction of TNF-alpha and NO by LPS stimulated macrophages can be markedly inhibited by quercetin, a major active component of plant Rhododendron cinnabarium.     

In vivo neutralization of tumor necrosis factor-alpha and interferon-gamma abrogates resistance to Yersinia enterocolitica infection in mice

Cytokines are important mediators of the inflammatory host response against infectious agents. In this study, the role of tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) in the elimination of a primary infection with highly virulent Yersinia enterocolitica serotype 0:8 strain WA-P has been investigated in C57BL/6 mice. The injection of anti-TNF- or anti-IFN- antibodies (serotherapy) prior to the intravenous challenge of a sublethal dose of Y. enterocolitica caused an increased bacterial net-growth in the spleens, although this effect was more pronounced for anti-TNF- treatment. The later treatment with anti-TNF- or anti-IFN- antibodies on day 3 post infection likewise abrogated resistance to Y. enterocolitica and, subsequently, led to death from progressive infection. Our data demonstrate for the first time that the endogenous production of both the cytokines TNF- and IFN- is required for the restriction of a primary Y. enterocolitica infection in mice.     

Tumor necrosis factor-alpha gene expression in human whole blood

Tumor necrosis factor-alpha (TNF) is recognized as a principal mediator of a variety of pathophysiologic and immunologic events. Lipopolysaccharide (LPS) challenge, either in vitro or in vivo, results in significant TNF production. In this study we present data demonstrating LPS-induced TNF mRNA expression and bioactivity using an in vitro tissue system of whole blood (WB). The kinetics of LPS-induced TNF production by WB was significantly accelerated as compared to isolated cultured peripheral blood monocytes (PBM). At post-LPS challenge, plasma from WB demonstrated a rapid rise in TNF bioactivity, peaking by 4 hr (1,021 units/ml/10(6) cells), plateauing between 4 and 8 hr, and then decreasing over the next 16 hr. In contrast, the highest measured TNF bioactivity from PBM did not occur until the 24-hr time-point (175 units/ml/10(6) cells). Whole blood buffy-coat TNF mRNA was assessed by Northern blot analysis, and demonstrated significant TNF mRNA accumulation at 1 hr and a peak 2 hr post-LPS challenge. By 8 hr TNF mRNA was undetectable. Concomitant administration of LPS with either prostaglandin E2 (10(-6)M) or Dexamethasone (10(-6)M) resulted in significant suppression of LPS-induced TNF production. This data supports WB as a useful in vitro medium for the molecular and cellular analysis of TNF. As specialized connective tissue, WB may provide an important environment to study the pharmacologic manipulation of TNF mRNA and bioactivity.     

Role of tumor necrosis factor alpha in induction of murine CD14 gene expression by lipopolysaccharide.

We previously demonstrated CD14 gene expression in myeloid and epithelial cells of the mouse and showed that expression of the CD14 gene in both is modulated by lipopolysaccharide (LPS). Here we test the hypothesis that the induction of CD14 in these cells is an indirect effect of LPS, one mediated by tumor necrosis factor alpha (TNF-alpha). TNF-alpha induced a transient increase in levels of CD14 in plasma with a peak at 6 to 8 h, and this increase in levels of CD14 antigen in plasma was accompanied by increased levels of CD14 mRNA in lung, liver, and kidney. Moreover, in situ hybridization studies revealed that CD14 mRNA was induced in both myeloid cells and epithelial cells, the same cells that respond to LPS. Pretreatment of mice with anti-TNF antiserum reduced the LPS-mediated increase in levels of CD14 in plasma and significantly reduced the level of induction of CD14 mRNA in selected epithelial cells in the kidney and liver. The antiserum did not appear to block LPS-mediated induction in myeloid cells in the tissues examined. In C3H/HeJ mice, the epithelial response to LPS was markedly attenuated whereas the response to TNF-alpha was normal. Thus, regulation of CD14 gene expression by LPS differs in epithelial and myeloid cells, with the epithelial responses in kidney and liver being mediated, in part, by TNF-alpha.     

In vitro tumor necrosis factor-alpha secretion by monocytes from patients with cystic fibrosis.

Immunoreactive tumor necrosis factor-alpha (TNF-alpha) concentration is increased in plasma from patients with cystic fibrosis and chronic Pseudomonas aeruginosa pulmonary infection. To determine if circulating monocytes could be the source of plasma TNF-alpha, we determined in vitro basal and endotoxin-stimulated TNF-alpha secretion by monocytes. In 10 adult patients studied at the time of a symptomatic respiratory exacerbation, basal secretion of TNF-alpha was significantly less than that for 10 matched healthy controls (median 265 pg/micrograms DNA, nonparametric 95% confidence interval 193 to 463 pg/micrograms DNA versus 575, 298 to 923 pg/micrograms DNA; P less than 0.006), although both groups responded equally effectively to added Escherichia coli endotoxin at greater than or equal to 25 ng/ml. In six patients and six matched controls, monocyte culture was repeated after completion of 2 wk anti-pseudomonal antibiotic treatment in the patients. The reduced basal TNF-alpha secretion in the patients had reversed and was not significantly different to that of controls. This effect mirrored a significant reduction in plasma immunoreactive TNF-alpha in these patients (mean +/- SD, 258 +/- 59.3 pg/ml pretreatment versus 133 +/- 47.8 pg/ml post-treatment; P less than 0.05). These findings suggest that a reversible downregulation of TNF-alpha secretion occurred at the time of a symptomatic respiratory deterioration in the presence of chronic P. aeruginosa infection. This may represent a physiologic regulatory mechanism to maintain a local inflammatory response to chronic pulmonary infection in cystic fibrosis.     

Dual role of tumor necrosis factor-alpha in angiogenesis.

The role of tumor necrosis factor-alpha (TNF; cachectin) in angiogenesis has been controversial. In vitro TNF inhibits proliferation of endothelial cells (EC) whereas in the cornea it appears to stimulate vessel growth. The authors tested TNF in their recently developed disc angiogenesis system (DAS), designed to measure the proliferation of microvessels. The DAS, implanted subcutaneously in mice, was either fitted with a central pellet containing mouse recombinant TNF (mrTNF), or exposed to mrTNF delivered subcutaneously by an osmotic minipump. Low doses of mrTNF (0.01-1 ng) induced angiogenesis, which was maximum at 0.1 ng, whereas high doses (1, and 5 micrograms) inhibited it. Subcutaneous mrTNF delivered at the (high) rate of 15-60 ng/hr for 14 days inhibited angiogenesis. These results indicate bimodal, dose-dependent opposing effects and explain some of the in vitro versus in vivo paradoxical results. TNF (native or exogenous) may have opposing effects on microvessels of neoplasms and inflammatory reactions, depending on its local tissue concentrations.     

Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha.

We evaluated the role of an endogenous production of nitric oxide (NO) in the in vitro migration of endothelial cells and in the in vivo angiogenic response elicited by platelet-activating factor (PAF), tumor necrosis factor-alpha (TNF), and basic fibroblast growth factor (bFGF). The NO synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME), but not its enantiomer D-NAME, prevented chemotaxis of endothelial cells induced in vitro by PAF and by TNF. The motogenic activity of TNF was also inhibited by WEB 2170, a specific PAF-receptor antagonist. In contrast, chemotaxis induced by bFGF was not prevented by L-NAME or by WEB 2170. Angiogenesis was studied in vivo in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model, the angiogenesis induced by PAF and TNF was inhibited by WEB 2170 and L-NAME but not by D-NAME. In contrast, angiogenesis induced by bFGF was not affected by L-NAME or by WEB 2170. TNF, but not bFGF, induced PAF synthesis within Matrigel. These results suggest that NO mediates the angiogenesis induced by PAF as well as that induced by TNF, which is dependent on the production of PAF. In contrast, the angiogenic effect of bFGF appears to be both PAF and NO independent.     

Tumor necrosis factor-alpha induces cell type and tissue-specific expression of chemoattractant cytokines in vivo.

Recombinant murine tumor necrosis factor-alpha (TNF-alpha) was shown to be a strong, systemic stimulus in vivo for members of the chemoattractant cytokine gene families (JE, KC, IP-10). The three genes showed differential sensitivity to TNF-alpha, and their expression demonstrated differential tissue specificity. IP-10 was the most strongly induced messenger RNA and was seen in the liver, kidney, and spleen but very poorly in the lung or skin. JE exhibited a similar pattern, though the magnitude of expression was markedly lower. KC expression was seen only in the liver of TNF-alpha-treated mice. The time course of expression for IP-10 was rapid and transient and showed strong dose dependence. In mice treated with TNF-alpha intravenously, messenger RNA was localized in the splenic stroma but not in adherent macrophages or nonadherent lymphocytes. In situ hybridization found the majority of intercrime expression in the splenic red pulp with little or no expression seen in the white pulp. In vitro, TNF-alpha was a potent stimulus of chemoattractant messenger RNA expression in fibroblasts but not in inflammatory peritoneal macrophages. These results indicate that TNF-alpha may be an important stimulus for chemoattractant cytokine gene expression in vivo, and the primary cell types responsible may be either stromal fibroblasts, microvascular endothelium, and/or a subset of anchored mononuclear phagocytes.     

Early expression of proinflammatory cytokines interleukin-1 and tumor necrosis factor-alpha after corneal transplantation.

This study's aim was to determine the early postoperative expression of proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) by corneal grafts. BALB/c (n = 90) and C57BL/6 (n = 90) murine recipients were grafted with donor corneas from either syngeneic or allogeneic mice. At 7 and 14 days after surgery, corneal grafts were excised and the recipient rims separated from the donor tissue. Corneal segments were cultured and assayed for cytokines by enzyme-linked immunosorbent assay (ELISA). There was profound upregulation in expression of both IL-1alpha and TNF-alpha after corneal transplantation. Among both low-rejecting BALB/c and high-rejecting C57BL/6 hosts, levels of IL-1alpha were significantly (p < 0.01) more marked in allogeneic as compared to syngeneic grafts. TNF-alpha overexpression was similarly more marked in allogeneic as compared to syngeneic grafts in both BALB/c and C57BL/6 hosts, although the difference was generally more marked among high-rejecting C57BL/6 recipients. In the case of both IL-1alpha and TNF-alpha, the principal source of cytokine expression in the transplanted tissue was the recipient rim. There is significant overexpression of both IL-1alpha and TNF-alpha during the first 2 weeks after transplantation in both syngeneic and allogeneic orthotopic corneal grafts. However, whereas in syngeneic grafts cytokine expression generally decreases after the first postoperative week, significantly elevated cytokine levels are sustained in allogeneic grafts, implicating IL-1 and TNF-alpha as mediators of the alloimmune response in corneal transplantation.     

Increased tumor necrosis factor-alpha (TNF-alpha) gene expression in parainfluenza type 1 (Sendai) virus-induced bronchiolar fibrosis.

Increased airway resistance and airway hyperresponsiveness induced in rats by infection with parainfluenza type I (Sendai) virus is associated with bronchiolar fibrosis. To determine whether increased tumor necrosis factor (TNF)-alpha gene expression is an important regulatory event in virus-induced bronchiolar fibrosis, pulmonary TNF-alpha mRNA and protein expression was assessed in rat strains that are susceptible (Brown Norway; BN) and resistant (Fischer 344; F344) to virus-induced bronchiolar fibrosis. Virus-inoculated BN rats had increased TNF-alpha pulmonary mRNA levels (P < 0.05) and increased numbers of bronchiolar macrophages and fibroblasts expressing TNF-alpha protein compared with virus-inoculated F344 rats (P < 0.05). Virus inoculation also induced elevated TNF-alpha mRNA and protein levels (P < 0.05) in cultured rat alveolar macrophages (NR8383 cells). A 55-kd soluble TNF receptor-immunoglobulin G fusion protein (sTNFR-IgG) was used to inhibit TNF-alpha bioactivity in virus-inoculated BN rats. Treated rats had fewer proliferating bronchiolar fibroblasts, as detected by bromodeoxyuridine incorporation, compared with virus-inoculated control rats (P < 0.05). There was also increased mortality in p55sTNFR-IgG-treated virus-inoculated rats associated with increased viral replication and decreased numbers of macrophages and lymphocytes in bronchoalveolar lavage fluid (P < 0.05). The results of this study indicate that 1) Sendai virus can directly up-regulate TNF-alpha mRNA and protein expression in macrophages, 2) TNF-alpha is an important mediator of virus-induced bronchiolar fibrosis, and 3) TNF-alpha has a critical role in the termination of Sendai viral replication in the lung.     

Effect of retinoids on the release and gene expression of tumor necrosis factor-alpha in human peripheral blood monocytes

The effect of retinoic acid (RA) and retinol (ROH) on the release of tumor necrosis factor (TNF) by human peripheral blood monocytes (HPBM) was determined. HPBM were cultured for various periods of time in either 5% complete (cAB) or delipidized (DLS) AB serum. TNF release (L929 cytolytic assay) in the presence of cAB occurred during the first 3 days of in vitro culture. Delipidization of AB serum completely inhibited the lipopolysaccharide (LPS)-induced release of TNF by HPBM. Addition of RA (0.5 microM) to DLS restored LPS-induced TNF release by HPBM, and supplementation with ROH (1.0 microM) resulted in release of TNF-like activity, but only after 3 days of in vitro culture. The maintenance of TNF release by the addition of exogenous RA after 3 days of in vitro culture suggested that depletion of endogenous RA was partially responsible for loss of TNF-like activity. The levels of endogenous TNF protein and mRNA were not influenced by delipidization of serum and were found to be similar to those of HPBM cultured in the presence of AB serum. TNF protein and mRNA were undetectable in HPBM ROH-treated cell lysates, although cytolytic activity was observed in culture supernatants. These results suggest that retinoids are required for the release of cytolytic factors from HPBM and that non-TNF cytolytic factors may be released by these cells at different stages of maturation.