Role of C5a in multiorgan failure during sepsis.

In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well known complication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model of sepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOF occurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressure of O(2), rising partial pressure of CO(2)). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis with increased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and to the bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but binding of fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatly attenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotactic responses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a prevents development of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects of anti-C5a in the CLP model of sepsis.

C-C chemokine-induced eosinophil chemotaxis during allergic airway inflammation.

The production of eosinophil-specific chemotactic factors during allergic airway responses may be a pivotal event resulting in eosinophil accumulation, activation, and airway damage. Recent studies have identified specific chemokines that may play crucial roles in recruitment of eosinophils to the site of allergic reactions. In this study we have utilized an established model of schistosome egg antigen (SEA) -mediated allergic responses to examine the role of specific C-C chemokines [macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemoattractant protein-1 (MCP-1)] in eosinophil recruitment. We have previously identified a role for MIP-1alpha in eosinophil accumulation in the lung and airway during allergic airway inflammation. We extend those studies using in vitro eosinophil chemotaxis to establish that both MIP-1alpha and RANTES are potent eosinophil chemotactic factors in lungs during allergic airway responses. Morphometric analysis demonstrated a peribronchial accumulation of eosinophils within the lungs beginning at 8 h, peaking at 24 h, and plateauing at 48-96 h after allergen (SEA) challenge. Utilizing whole-lung homogenates from allergen-challenged mice, in vitro eosinophil chemotactic assays demonstrated significant increases in eosinophil chemotactic activity with 8-h lung homogenates and peak activity with samples from 24-h lung homogenates. These data correlated with the morphometric analysis of peribronchial eosinophil accumulation in situ. When lung homogenates from allergen-challenged mice were preincubated in vitro with antibodies specific for MIP-1alpha, RANTES, or MCP-1, a significant reduction in eosinophil chemotaxis was observed with only MIP-1alpha and RANTES neutralization. Altogether, these studies indicate that RANTES and MIP-1alpha are major eosinophil chemotactic factors produced during allergic airway responses.

The role of cytokine networks in the local liver injury following hepatic ischemia/reperfusion in the rat.

The liver is highly susceptible to a number of pathological insults, including ischemia/reperfusion injury. We have previously employed an animal model of hepatic ischemia/reperfusion injury, and have shown that this injury induces the production and release of hepatic-derived tumor necrosis factor alpha (TNF-alpha), which mediates, in part, local liver injury following hepatic reperfusion. In the present study, we have extended these previous observations to assess whether an interrelationship exists between TNF-alpha and the neutrophil chemoattractant/activating factor, epithelial neutrophil activating protein, that may account for some of the pathology of neutrophil-mediated ischemia/reperfusion-induced liver injury. We observed that hepatic ischemia/reperfusion injury leads to: (1) a coincident increase in hepatic neutrophil sequestration, elevated serum alanine aminotransferase (ALT) levels, and hepatic production of epithelial neutrophil activating protein; (2) passive immunization with neutralizing antibodies to TNF-alpha resulted in significant suppression of hepatic-derived epithelial neutrophil activating protein; and (3) neutralization of epithelial neutrophil activating protein by passive immunization significantly attenuated neutrophil sequestration in the liver and serum ALT levels. These findings support the notion that local expression of hepatic epithelial neutrophil activating protein produced in response to TNF-alpha is an important mediator of the local neutrophil-dependent hepatic injury associated with hepatic ischemia/reperfusion.

The role of stem cell factor (c-kit ligand) and inflammatory cytokines in pulmonary mast cell activation.

Mast cells play a critical role in allergic airway responses via IgE-specific activation and release of potent inflammatory mediators. In the present study, we have isolated and characterized primary mast cell lines derived from the upper airways of normal mice. The primary mast cell lines were grown and maintained by incubation with interleukin-3 (IL-3) and stem cell factor (SCF) and shown to be c-kit (SCF receptor) positive by flow cytometry. Subsequently, we examined the proliferation of both airway and bone marrow derived mast cell lines in response to inflammatory and hematopoietic cytokines, including SCF, IL-1, IL-3, interferon-gamma, IL-4, and IL-10. The results from the pulmonary mast cell lines were compared with those from bone marrow derived mast cells. Pulmonary mast cell lines were capable of proliferating in response to IL-3, IL-4, IL-10, and SCF, whereas the combination of SCF with the other cytokines did not increase the response over SCF alone. In contrast, the bone marrow-derived mast cells proliferated strongest to SCF or IL-3, but only modestly to IL-4 and IL-10. Furthermore, the combination of SCF with IL-3, but not the other cytokines, exhibited an increase in bone marrow-derived mast cell proliferation. Cytokine-specific stimulation of histamine release in the airway-derived and bone marrow-derived mast cells showed parallel results. SCF was the only cytokine shown to induce substantial histamine release. However, when certain nonhistamine releasing cytokines were combined with SCF, a synergistic increase in histamine release was induced in upper airway, but not bone marrow-derived mast cells. The results of these studies suggest that cytokines differentially modulate induction of proliferation and degranulation of bone marrow and upper airway-derived mast cells and may further indicate a cytokine activational cascade in tissue mast cells.

Age-dependent injury in human umbilical vein endothelial cells: relationship to apoptosis and correlation with a lack of A20 expression.

BACKGROUND: It has recently been shown that human umbilical vein endothelial cells (HUVEC) become increasingly sensitive to growth factor deprivation, resulting in cell death, as a function of age in culture. The overall goal of the present study was to investigate the mechanism of lethal injury in these cells and compare the injury process to other known mechanisms of injury in the same cells. EXPERIMENTAL DESIGN: HUVEC were established in culture and maintained for four passages. Injury to first-passage cells and fourth-passage cells were examined for injury in the presence of agents that are known to confer resistance to apoptosis. Ultrastructural features of injury and DNA fragmentation patterns were assessed. Expression of factors that are known to be associated with resistance to apoptosis in other models were assessed. RESULTS: Fourth-passage HUVEC undergoing injury exhibited morphologic features characteristic of apoptosis and DNA fragmentation. Agents known to inhibit apoptotic cell injury in other models inhibited injury. A20 expression was correlated with resistance to injury in fourth-passage HUVEC, but there was no correlation between bcl-2 and bcl-x expression and resistance to injury. CONCLUSIONS: HUVEC injury resulting from growth factor deprivation increases as a function of age in vitro and appears to be a form of apoptosis. A20 expression may confer resistance to cell injury through this pathway.

Macrophage inflammatory protein-1 alpha mediates lung leukocyte recruitment, lung capillary leak, and early mortality in murine endotoxemia.

Systemic exposure to LPS initiates a complex sequence of events resulting in organ-specific leukocyte recruitment and end-organ injury. We hypothesized that macrophage inflammatory protein-1 alpha (MIP-1 alpha), a C-C chemokine with leukocyte chemotactic and activating properties, may play an important role in lung inflammatory cell recruitment, subsequent lung injury, and mortality in endotoxemia. CD-1 mice were challenged with LPS (200 micrograms), resulting in a maximal 3.5-fold increase in neutrophils (polymorphonuclear leukocytes (PMNs)) at 6 h post-LPS, and a 2.6-fold increase in numbers of macrophages (M phi) within lung minces at 24 h. A time-dependent increase in MIP-1 alpha mRNA and protein was detected in lung after LPS treatment, with immunolocalization of MIP-1 alpha to blood and lung M phi, and the subendothelium. The pretreatment of mice with rabbit anti-MIP-1 alpha Ab resulted in a decrease in the influx of PMNs at 6 h, and influx of M phi at 24 h post-LPS challenge, an approximately 65% reduction in LPS-induced lung permeability to Evans blue, and a modest decrease in mortality at 24, but not 48 h post-LPS. Furthermore, passive immunization of mice with anti-MIP-1 alpha serum resulted in a 35% reduction in ICAM-1 mRNA levels within lung homogenates post-LPS. Finally, the pretreatment of animals with sTNFR:Fc (soluble TNF receptor:Ig construct) resulted in a 60% reduction in LPS-induced MIP-1 alpha mRNA expression within lung homogenates at 4 h post-LPS. Our studies indicate that MIP-1 alpha plays an integral role as a mediator of both PMN and M phi recruitment in murine endotoxemia.

Chemokine expression during hepatic ischemia/reperfusion-induced lung injury in the rat. The role of epithelial neutrophil activating protein.

The liver is highly susceptible to a number of pathological insults, including ischemia/reperfusion injury. One of the striking consequences of liver injury is the associated pulmonary dysfunction that may be related to the release of hepatic-derived cytokines. We have previously employed an animal model of hepatic ischemia/reperfusion injury, and demonstrated that this injury causes the production and release of hepatic-derived TNF, which mediates a neutrophil-dependent pulmonary microvascular injury. In this study, we have extended these previous observations to assess whether an interrelationship between TNF and the neutrophil chemoattractant/activating factor, epithelial neutrophil activating protein-78 (ENA-78), exists that may be accountable for the pathology of lung injury found in this model. In the context of hepatic ischemia/reperfusion injury, we demonstrated the following alterations in lung pathophysiology: (a) an increase in pulmonary microvascular permeability, lung neutrophil sequestration, and production of pulmonary-derived ENA-78; (b) passive immunization with neutralizing TNF antiserum resulted in a significant suppression of pulmonary-derived ENA-78; and (c) passive immunization with neutralizing ENA-78 antiserum resulted in a significant attenuation of pulmonary neutrophil sequestration and microvascular permeability similar to our previous studies with anti-TNF. These findings support the notion that pulmonary ENA-78 produced in response to hepatic-derived TNF is an important mediator of lung injury.

Immune complex vasculitis with secondary ulcerative dermatitis in aged C57BL/6NNia mice.

A spontaneous, severely pruritic ulcerative dermatitis was initially observed in 33/201 (16.4%) aged C57BL/6NNia mice obtained from the National Institute of Aging. This ulcerative dermatitis also developed in 21/98 (21%) aged C57BL/6 mice in a subsequent experimental group obtained from the same source. The average age of onset in the initial group was 20 months. These animals were negative for ectoparasite infestation and primary bacterial or fungal infection. The lesions varied from acute epidermal excoriation and ulceration to chronic ulceration with marked dermal fibrosis. In the affected animals, leukocytoclastic vasculitis was present in the dermis in both areas of ulceration and areas covered by normal intact epidermis. Immunofluorescent staining of the skin was positive for deposition of IgG, IgM, and fibrinogen in the dermal vessels of the affected mice. Leukocytoclastic vasculitis was not observed in unaffected animals, nor were deposits of immunoglobulin or fibrinogen present in the skin of the control animals. This study provides strong evidence that the ulcerative dermatitis is caused by an immune complex-induced vasculitis. The elucidation of the pathogenesis of this disease is important because of the significant percentage of animals affected and because the C57BL/6 mouse may be a useful model to study human vasculitides.

Mechanisms of hyperacute rejection in porcine liver transplantation. Antibody-mediated endothelial injury.

Hyperacute rejection after OLT is an unusual event. We have demonstrated previously that hepatic hyperacute rejection can occur in the presence of high titers of donor-specific cytotoxic antibody. This study addresses the issues of antibody localization within the allograft and the consequences of antibody deposition and complement activation within the transplanted organ. A porcine model of liver transplantation was used and 3 experimental groups were studied. Group I recipients (n = 6) were specifically sensitized to their liver donors with skin grafts from their liver donors before hepatic grafting. Group II recipients (n = 6) underwent third-party skin graft sensitization before liver transplantation. Group III recipients (n = 6) underwent liver grafting without prior sensitization. After liver transplantation, serum complement (CH50) levels declined promptly in all groups; a statistically significant drop as compared with control animals was seen only in group I (P < 0.05). Liver biopsies from donor-specific sensitized recipients showed massive injury by light microscopy within 30 min of revascularization, demonstrating fibrinoid necrosis of vessels and neutrophil influx. On immunofluorescent examination, liver specimens from donor-specific sensitized animals showed intense IgG, IgM, and C3 deposition in the vessels of the portal triads. Antibody deposition was not seen in third-party sensitized animals or control animals. On electron microscopy, control animals and third-party sensitized animals showed minimal ultrastructural alterations. In comparison, livers from donor-specific sensitized animals showed severe microvascular injury with destruction of endothelial cells, edema, hemorrhage, and hepatocyte necrosis. In a passive serum transfer experiment carried out by infusing serum from a skin graft-sensitized pig directly into the portal vein of the skin graft donor, severe liver injury was evident, with identification of antibody deposition by light and electron microscopy and by immunofluorescence. These studies demonstrate that tissue injury in hyperacute hepatic rejection is mediated by antibody deposition within the grafted liver and is associated with systemic activation of the complement cascade.

Injury of rat pulmonary alveolar epithelial cells by H2O2: dependence on phenotype and catalase.

In a variety of inflammatory lung diseases, type I alveolar epithelial cells are more likely to be injured than are type II cells. Because oxidants have been implicated as a cause of injury in various inflammatory lung diseases, we evaluated the effects of differentiation on alveolar epithelial cell susceptibility to H2O2-induced injury. With the use of isolated rat type II cells in culture, we found that the cytotoxic effect of H2O2 increased between days 2 and 7, when type II cells are known to lose their distinctive type II properties and assume a more type I-like appearance. We previously reported that type II cells utilized both intracellular catalase and glutathione-dependent reactions to protect against H2O2. We therefore examined whether alterations in either of these protective mechanisms were responsible for the differentiation-dependent changes in sensitivity to H2O2. We found that catalase activity within alveolar epithelial cells decreased between 2 and 7 days in culture, whereas no changes were detected in glutathione-dependent systems. We then used a histochemical technique that detects catalase activity and found that type II cells within rat lungs possessed numerous catalase-containing peroxisomes, whereas very few were detected in type I cells. These findings demonstrate that as type II cells assume a type I-like phenotype, they become more susceptible to H2O2-induced injury. This increased susceptibility is associated with reductions in intracellular catalase activity, both in vitro and in vivo.

Experimental obliterative cholangitis. A model for the study of biliary atresia.

Noninfectious obliterative cholangitis results from biliary tract inflammation in clinical conditions such as biliary atresia and sclerosing cholangitis. The purpose of this study was to develop an animal model of noninfectious biliary tract inflammation and fibrosis. An implantable osmotic pump was connected to a catheter placed into the gallbladder of hamsters. Phorbol myristate acetate (PMA) was infused into the biliary tract for periods of 6 hours to 28 days. After 7 days the animals developed neutrophil infiltration, cellular necrosis, and edema of the biliary ducts. After 14 days, the animals demonstrated intrahepatic cholestasis with bile duct fibrosis and acute and chronic inflammatory cell infiltration. By 28 days pronounced portal fibrosis was present, some of which created an early bridging cirrhosis pattern. In addition there was evidence of neocholangiogenesis. We conclude that long-term PMA infusion into the biliary tract generates an inflammatory response characterized by obliterative cholangitis and fibrosis, sharing many of the histologic features of human biliary atresia. This model may provide a relatively simple technique for investigating the process of nonpyogenic biliary tract inflammation.

Tumor necrosis factor participates in the pathogenesis of acute immune complex alveolitis in the rat.

We have examined the role of intrapulmonary TNF in a rat model of acute immune complex-triggered alveolitis. Intratracheal instillation of IgG anti-bovine serum albumin (anti-BSA) followed by intravenous infusion of BSA results in acute alveolitis. Over the 4-h course of evolving lung injury, a 10-fold increase in TNF activity occurred in bronchoalveolar lavage (BAL) fluid. Immunohistochemical analysis of lung sections and BAL cells revealed that alveolar macrophages are the chief source of TNF. Antibodies that specifically neutralize rat TNF activity were raised in rabbits immunized with recombinant mouse TNF alpha. When administered into the lungs with anti-BSA, anti-TNF resulted in a marked reduction (up to 61%) in lung injury. Intratracheal instillation of exogenous TNF alone, or in combination with anti-BSA, resulted in an increase in lung injury compared to controls. Morphometric analysis and measurements of myeloperoxidase activities in whole lung extracts from rats treated with anti-TNF revealed a marked reduction in neutrophils compared to positive controls. The anti-TNF antibody preparation did not inhibit in vitro complement activation or diminish neutrophil chemotactic activity present in activated rat serum. These data indicate that intrapulmonary TNF activity is required for the full development of acute immune complex-triggered alveolitis, that alveolar macrophages are the primary source of this cytokine, and that TNF participates in the pathogenesis of immune complex alveolitis through a mechanism involving neutrophil recruitment.

Acute acid aspiration lung injury in the rat: biphasic pathogenesis.

The purpose of this study was to develop a reproducible model of acute acid aspiration-induced lung injury in the rat to explore the pathophysiology of aspiration pneumonitis. A biphasic injury pattern was observed with injury peaks at 1 hr and 4 hr. Histologic studies at 4 hr revealed significant increases in neutrophils in the alveolar interstitial space. These studies suggest that acid aspiration results in a biphasic acute injury. We hypothesize that the first phase results from a direct physiochemical process or is mediated via afferent (capsaicin sensitive) nerves or both. The second phase, occurring 2-3 hr later, is mediated by neutrophils and is consistent with an acute inflammatory response.

Ultrastructural cytochemical analysis of oxygen radical-mediated immunoglobulin A immune complex induced lung injury in the rat.

Recent studies suggest that IgA immune complex induced lung injury in the rat is oxygen radical mediated. A cerium chloride (CeCl3) method was used to ultrastructurally analyze the in situ elaboration of H2O2 in IgA immune complex injured lungs. After induction of IgA immune complex lung injury, the lungs were instilled with a reaction buffer containing CeCl3 which forms an electron-dense precipitate when exposed to H2O2. Ultrastructural examination and x-ray microanalysis revealed electron-dense cerium deposits on the surfaces and in cytoplasmic vacuoles of alveolar macrophages located along damaged alveolar septae. Similar deposits were prominent on the luminal surfaces of injured pneumocytes, especially alveolar type II cells. No cerium-containing deposits were found in undamaged negative control lungs (IgA alone without antigen) or in lungs of rats that received IgA immune complexes in the presence of catalase. To further define the source of cerium-reactive products, monolayers of rat pulmonary alveolar epithelial cells were incubated with IgA complexes. Alveolar epithelial cells exposed to complexes produced no detectable H2O2 as measured by two spectrophotometric assays, and in the presence of CeCl3, exhibited negligible amounts of electron-dense material regardless of the presence or absence of catalase. The data corroborate indirect in vivo and in vitro studies which suggest that IgA immune complex induced lung injury is mediated by oxygen-derived metabolites produced by lung macrophages. Use of the CeCl3 method in intact rat lungs allows direct ultrastructural cytochemical analysis of H2O2 production in inflamed tissue.

Pancreatitis-induced acute lung injury. An ARDS model.

Cerulein-induced acute pancreatitis in rats is associated with acute lung injury characterized by increased pulmonary microvascular permeability, increased wet lung weights, and histologic features of alveolar capillary endothelial cell and pulmonary parenchymal injury. The alveolar capillary permeability index is increased 1.8-fold after a 3-hour injury (0.30 to 0.54, p less than 0.05). Gravimetric analysis shows a similar 1.5-fold increase in wet lung weights at 3 hours (0.35% vs. 0.51% of total body weight, p less than 0.05). Histologic features assessed by quantitative morphometric analysis include significant intra-alveolar hemorrhage (0.57 +/- 0.08 vs. 0.12 +/- 0.02 RBC/alveolus at 6 hours, p less than 0.001); endothelial cell disruption (28.11% vs. 4.3%, p less than 0.001); and marked, early neutrophil infiltration (7.45 +/- 0.53 vs. 0.83 +/- 0.18 PMN/hpf at 3 hours, p less than 0.001). The cerulein peptide itself, a cholecystokinin (CCK) analog, is naturally occurring and is not toxic and in several in vitro settings including exposure to pulmonary artery endothelial cells, Type II epithelial cells, and an ex vivo perfused lung preparation. The occurrence of this ARDS-like acute lung injury with acute pancreatitis provides an excellent experimental model to investigate mechanisms and mediators involved in the pathogenesis of ARDS.

Phagocytosis of Candida albicans enhances malignant behavior of murine tumor cells.

Murine tumor cells were induced to phagocytize either Candida albicans or group A streptococcal cells. The presence of microbial particles within the tumor cell cytoplasm had no effect on in vitro tumor cell growth. However, when Candida albicans-infected tumor cells were injected into syngeneic mice, they formed tumors that grew faster, invaded the surrounding normal tissue more rapidly and metastasized more rapidly than control tumor cells. Tumor cells infected with group A streptococcal particles did not grow faster or show increased malignant behavior. These data indicate that the in vivo behavior of malignant tumor cells can be modulated by microbial particles, which are often present in the microenvironment of the growing tumor.

Phorbol myristate acetate produces injury to isolated rat lungs in the presence and absence of perfused neutrophils.

Because lung injury induced by phorbol myristate acetate (PMA) has been reported by some to be mediated by blood neutrophils (PMN) and by others to occur independently of PMN, we examined the PMN dependency of PMA-induced injury in isolated, perfused lungs of rats. Depending on dose, PMA added to medium perfusing isolated rat lungs produced injury in the presence or in the absence of added PMN. When a high concentration of PMA (57 ng/ml) was added to medium devoid of added PMN, perfusion pressure and lung weight increased. Superoxide dismutase (500 U/ml) and catalase (400 U/ml) added to the medium prior to PMA had no effect on the increases in lung weight or perfusion pressure. When a concentration of PMA (21 ng/ml or less) that did not by itself cause lungs to accumulate fluid was added to perfusion medium containing PMN (1 X 10(8)), superoxide was produced, perfusion pressure increased, and lungs accumulated fluid. Addition of superoxide dismutase and catalase to this preparation prevented the increase in lung weight, but not the increase in perfusion pressure. We conclude that high concentrations of PMA produce lung injury which is independent of neutrophils and oxygen radicals and that lower concentrations produce injury which is neutrophil-dependent and mediated by oxygen radicals. These results may explain why PMA-induced lung injury has variously been reported to be PMN-dependent in some systems and PMN-independent in others.

Comparative O2-. responses of lung macrophages and blood phagocytic cells in the rat. Possible relevance to IgA immune complex induced lung injury.

IgA immune complex-induced lung injury in the rat is oxygen radical mediated and partially complement-dependent but develops fully after neutrophil depletion. The extent to which monocytes, lung interstitial macrophages, and alveolar macrophages may be involved in the development of lung injury in this model is unclear. To further understand the pathogenesis of IgA lung injury, we have examined the capacity of phagocytic cells isolated from different anatomic compartments of the lung to produce toxic oxygen-derived metabolites. [3H]Thymidine pulse labeling and autoradiography as well as in vivo phagocytosis studies were used to distinguish macrophages isolated from the alveolar and interstitial compartments. Lung interstitial macrophages were characterized ultrastructurally, cytochemically, and functionally. Interstitial macrophages were relatively uniform in size, had blunt pseudopodia, and contained almost no intracytoplasmic lamellar inclusions compared to alveolar macrophages. Similar to monocytes and alveolar macrophages, interstitial macrophages contained nonspecific esterase activity and exhibited the capacity to phagocytize latex and opsonized zymosan particles. Lung interstitial and alveolar macrophages incubated with IgA immune complexes, IgG immune complexes, or phorbol ester (PMA) produced similar amounts of O2-. in a dose-dependent manner. In contrast, peripheral blood neutrophils responded to IgG immune complexes and PMA but not to IgA immune complexes. Monocytes produced a small amount of O2-. in response to PMA but almost no O2-. in response to IgA or IgG immune complexes. These data are consistent with recent in vivo studies which indicate that IgA immune complex lung injury is neutrophil independent. The data provide direct in vitro evidence that lung interstitial and alveolar macrophages produce O2-. following incubation with PMA, IgA, or IgG immune complexes and may therefore contribute to the development of oxygen radical mediated lung injury.

Acute in vivo effects of human recombinant tumor necrosis factor.

Tumor necrosis factor is a peptide cytokine that induces hemorrhagic necrosis of some tumors and is responsible for the severe cachexia observed in advanced infectious diseases. We evaluated the acute effects of intravenous administration of purified human recombinant tumor necrosis factor in mice. With as little as 0.01 microgram/mouse (0.00045 mg/kg) a peripheral blood lymphopenia and neutrophilia developed as determined by flow cytometric analysis. At 1 microgram/mouse, the lymphopenia was both relative (21 +/- 3% versus 65 +/- 3%; p less than 0.001 treated versus control) and absolute (62 +/- 10 versus 229 +/- 29 X 10(4) cells/ml p less than 0.001). The neutrophilia was also relative (79 +/- 3% versus 34 +/- 3%; p less than 0.001 treated versus control) and absolute (237 +/- 26 versus 110 +/- 13 X 10(4) cell/ml; p less than 0.001). The neutrophilia was due to an increase in both mature and immature cells. At the higher doses the animals developed hypovolemic shock with an increased hematocrit and watery diarrhea occurred. Microscopic examination of the small bowel disclosed necrosis of the villi. Ultrastructural studies of the small bowel confirmed the necrosis and also showed severe endothelial cell damage, pyknotic nuclei, exocytosis of Paneth cell granules, and extravasation of red blood cells and neutrophils into the interstitium. A vascular leak syndrome developed with preferential fluid loss into the small and large bowel. These data demonstrate the potent in vivo effects of purified human recombinant tumor necrosis factor.

Pulmonary alveolar macrophage function during acute inflammatory lung injury.

Pulmonary alveolar macrophages (PAM) are present during acute lung inflammation, yet the functional role of these cells in both the initiation and resolution of lung injury is not well defined. To better understand the relationship between PAM functional responses and the evolution of acute reversible lung injury, we examined the ability of both unstimulated and stimulated (PMA, zymosan) PAM to secrete reactive oxygen metabolites (superoxide anion O2-) and lysosomal enzymes (lysozyme, N-acetyl-B-D-glucosaminidase) at specific time points (0, 6, 12, 24, 48, and 72 h) after initiation of acute lung injury via reverse passive Arthus reaction in pathogen-free Sprague-Dawley rats. After acute lung injury, stimulated PAM produced increasing amounts of O2- compared with PAM from noninjured lungs. Maximal O2- production by PAM occurred at 24 h after lung injury, at which time a 3.5-fold and 50% increase in O2- production by PAM was observed when PAM were stimulated with PMA and zymosan, respectively. The amount of O2- generated by these cells slowly decreased during the next 48 h. Enhanced generation of O2- by PAM from injured lungs was not due to altered enzymatic activity of the O2--producing NADPH oxidase, nor was it due to an absolute increase in the NADPH oxidase in "activated" PAM. These observations suggest that increased O2- generation by PAM from injured lungs is due to enhancement of mechanisms responsible for induction of oxidase activity. In addition, a differential accumulation and secretion of lysozyme and N-acetyl-B-D-glucosaminidase activity by PAM was observed after acute lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)