Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa

According to the widely accepted view, neutrophil elastase (NE), a neutrophil-specific serine protease, is a major contributor to Pseudomonas aeruginosa infection-associated host tissue inflammation and damage, which in severe cases can lead to death. Herein, we provide for the first time compelling evidence that the host rather employs NE to protect itself against P. aeruginosa infection. Using a clinically relevant model of pneumonia, targeted deficiency in NE increased the susceptibility of mice to P. aeruginosa. We found that NE was required for maximal intracellular killing of P. aeruginosa by neutrophils. In investigating the mechanism of NE-mediated killing of P. aeruginosa, we found that NE degraded the major outer membrane protein F, a protein with important functions, including porin activity, maintenance of structural integrity, and sensing of host immune system activation. Consistent with this, the use of an isogenic mutant deficient in outer membrane protein F negated the role of NE in host defense against P. aeruginosa infection.     

Elastase inhibitors]

The serine proteinase elastase is located in the azurophil granules of mature circulating polymorphonuclear neutrophils. This neutrophil elastase or NE is a potent non specific serine protease which plays a role as bactericidal agent and in the degradation of immune complexes by intraphagosomal processes. It promotes inflammation when the granule contents are secreted in the extracellular environment. In certain pathological circumstances, an imbalance between NE and its major plasmatic inhibitor alpha 1-PI (formerly, alpha 1-antitrypsin) leads to abnormal tissue destruction and disease development. Genetic or acquired alpha 1-PI deficiency is thought to be involved in the pathogenesis of pulmonary emphysema. A variety of degenerative and degradative disorders are also associated to uncontrolled proteolysis by NE (rheumatoid arthritis, glomerulonephritis, adult respiratory distress symptom, psoriasis, cancer). Numerous inhibitors of NE have been reported. Various molecules are currently undergoing clinical trials for emphysema and other pulmonary diseases.     

Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5-Lys-6 and Val-9-Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6-Gln-57 and Ser-10-Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.     

Red blood cells increase secretion of matrix metalloproteinases from human lung fibroblasts in vitro

Tissue remodeling is an important process in many inflammatory and fibrotic lung disorders. RBC may in these conditions interact with extracellular matrix (ECM). Fibroblasts can produce and secrete matrix components, matrix-degrading enzymes (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Imbalance in matrix synthesis/degradation may result in rearrangement of tissue architecture and lead to diseases such as emphysema or fibrosis. Neutrophil elastase (NE), a protease released by neutrophils, is known to activate MMP. We hypothesized that RBC can stimulate secretion of MMPs from human lung fibroblasts and that NE can augment this effect. Human fetal lung fibroblasts were cultured in floating collagen gels with or without RBC. After 4 days, the culture medium was analyzed with gelatin zymography, Western blot, and ELISA for MMP-1, -2, -3 and TIMP-1, -2. RBC augmented NE-induced fibroblast-mediated collagen gel contraction compared with NE alone (18.4+/-1.6%, 23.7+/-1.4% of initial gel area, respectively). A pan-MMP inhibitor (GM-6001) completely abolished the stimulating effect of NE. Gelatin zymography showed that RBC stimulated MMP-2 activity and that NE enhanced conversion to the active form. Addition of GM-6001 completely inhibited MMP-2 activity in controls, whereas it only partially altered RBC-induced MMP activity. Western blot confirmed the presence of MMP-1 and MMP-3 in fibroblasts stimulated with RBC, and ELISA confirmed increased concentrations of pro-MMP-1. We conclude that stimulation of MMP secretion by fibroblasts may explain the ability of RBC to augment fibroblast-mediated collagen gel contraction. This might be a potential mechanism by which hemorrhage in inflammatory conditions leads to ECM remodeling.     

SERPINB1 upregulation is associated with in vivo complex formation with neutrophil elastase and cathepsin G in a baboon model of bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) continues to be a major cause of morbidity in premature infants. An imbalance between neutrophil elastase and its inhibitors has been implicated in BPD. Serine protease inhibitor (SERPIN)B1 is an inhibitor of neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (cat G). Recent studies suggest that SERPINB1 could provide protection in the airways by regulating excess protease activity associated with inflammatory lung disorders. In this study, we determined the distribution and ontogeny of SERPINB1 in the baboon lung and characterized the expression of SERPINB1 in baboon models of BPD. SERPINB1 expression was detected in the conducting airway and glandular epithelial cells in addition to neutrophils, macrophages, and mast cells. SERPINB1 mRNA and protein expression increased with advancing gestational age and in the new BPD model. In contrast, SERPINB1 expression levels were decreased in the old BPD model. Furthermore, SERPINB1 was detected as a high-molecular-mass (HMM) complex in lung tissue and bronchoalveolar lavage fluid samples from the BPD group. Analysis of the HMM complex by coimmunoprecipitation showed that these complexes were formed between SERPINB1 and NE or cat G. High-performance liquid chromatography (HPLC) ion trap mass spectrometry verified the presence of SERPINB1 in HMM complexes. Finally, NE activity level was compared between new and old baboon models of BPD and was found to be significantly lower in new BPD. Thus SERPINB1 upregulation in new BPD may be protective by contributing to the regulation of neutrophil proteases NE and cat G.     

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.     

Adenoviral augmentation of elafin protects the lung against acute injury mediated by activated neutrophils and bacterial infection

During acute pulmonary infection, tissue injury may be secondary to the effects of bacterial products or to the effects of the host inflammatory response. An attractive strategy for tissue protection in this setting would combine antimicrobial activity with inhibition of human neutrophil elastase (HNE), a key effector of neutrophil-mediated tissue injury. We postulated that genetic augmentation of elafin (an endogenous inhibitor of HNE with intrinsic antimicrobial activity) could protect the lung against acute inflammatory injury without detriment to host defense. A replication-deficient adenovirus encoding elafin cDNA significantly protected A549 cells against the injurious effects of both HNE and whole activated human neutrophils in vitro. Intratracheal replication-deficient adenovirus encoding elafin cDNA significantly protected murine lungs against injury mediated by Pseudomonas aeruginosa in vivo. Genetic augmentation of elafin therefore has the capacity to protect the lung against the injurious effects of both bacterial pathogens resistant to conventional antibiotics and activated neutrophils.     

Administration of a synthetic antiprotease reduces smoke-induced lung injury.

Our previous studies suggest that a neutrophil-mediated inflammatory injury causes a major fraction of the pulmonary edema that occurs after smoke inhalation. Because activated neutrophils extrude cytotoxic proteases, the current study was conducted to evaluate the role of proteases in the pulmonary microvascular injury. Twelve sheep, instrumented for collection of lung lymph, were insufflated with cotton smoke. The sheep were treated 30 min after smoke inhalation with either gabexate mesilate (an inhibitor of serine proteases) or vehicle. Smoke inhalation resulted in an increased protease activity in the lung interstitium, as evidenced by decreases in both antiprotease activity and immunoreactive alpha 2-macroglobulin. Intravenous infusion of gabexate mesilate prevented the decrease in antiprotease activity. The protease inhibitor significantly attenuated the smoke-induced increase in transvascular fluid and protein flux, with untreated animals exhibiting 460% increases in flux compared with 180% in the inhibitor treated sheep. The protease inhibitor also eliminated the functional degradation in gas exchange that was observed in the untreated sheep. These studies strongly suggest that an increase in pulmonary proteolytic enzyme activity is responsible for a significant fraction of the degradation in microvascular integrity and gas exchange that is associated with smoke inhalation injury.     

The pathophysiology of superoxide: roles in inflammation and ischemia

The superoxide radical plays major roles in the neutrophil-medicated acute inflammatory response and in postischemic tissue injury, although the sources and actions of the radical are quite different in these two pathological states. While neutrophils produce superoxide for the primary purpose of aiding in the killing of ingested microbes, a second useful function has evolved. The superoxide released from actively phagocytosing neutrophils serves to attract more neutrophils by reacting with, and activating, a latent chemotactic factor present in plasma. Superoxide dismutase, by preventing the activation of this superoxide-dependent chemotactic factor, exerts potent anti-inflammatory action. During ischemia, energy-starved tissues catabolize ATP to hypoxanthine. Calcium transients in these cells appear to activate a calmodulin regulated protease which attacks the enzyme xanthine dehydrogenase, converting it to a xanthine oxidase capable of superoxide generation. When the tissue is reperfused and reoxygenated, all the necessary components are present (xanthine oxidase, hypoxanthine, and oxygen) to produce a burst of superoxide which results in extensive tissue damage. Ischemic tissues are protected by superoxide dismutase or allupurinol, an inhibitor of xanthine oxidase.     

Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity

After bacterial infection, neutrophils dominate the cellular infiltrate. Their main function is assumed to be killing invading pathogens and resolving the inflammation they cause. Activated neutrophils are also known to release a variety of molecules, including the neutrophil serine proteinases, extracellularly. The release of these proteinases during inflammation creates a proteolytic environment where degradation of different molecules modulates the inflammatory response. Flagellin, the structural component of flagella on many bacterial species, is a virulence factor with a strong proinflammatory activity on epithelial cells and other cell types. In this study we show that both human and mouse neutrophil serine proteinases cleave flagellin from Pseudomonas aeruginosa and other bacterial species. More important, cleavage of P. aeruginosa flagellin by the neutrophil serine proteinases neutrophil elastase and cathepsin G resulted in loss of the biological activity of this virulence factor, as evidenced by the lack of innate host defense gene expression in human epithelial cells. The finding that flagellin is susceptible to cleavage by neutrophil serine proteinases suggests a novel role for these enzymes in the inflammatory response to infection. Not only can these enzymes kill bacteria, but they also degrade their virulence factors to halt the inflammatory response they trigger.     

Inhibition of neutrophil elastase by alpha1-protease inhibitor at the surface of human polymorphonuclear neutrophils

The uncontrolled proteolytic activity in lung secretions during lung inflammatory diseases might be due to the resistance of membrane-bound proteases to inhibition. We have used a new fluorogenic neutrophil elastase substrate to measure the activity of free and membrane-bound human neutrophil elastase (HNE) in the presence of alpha1-protease inhibitor (alpha1-Pi), the main physiological inhibitor of neutrophil serine proteases in lung secretions. Fixed and unfixed neutrophils bore the same amounts of active HNE at their surface. However, the HNE bound to the surface of unfixed neutrophils was fully inhibited by stoichiometric amounts of alpha1-Pi, unlike that of fixed neutrophils. The rate of inhibition of HNE bound to the surface of unfixed neutrophils was the same as that of free HNE. In the presence of alpha1-Pi, membrane-bound elastase is almost entirely removed from the unfixed neutrophil membrane to form soluble irreversible complexes. This was confirmed by flow cytometry using an anti-HNE mAb. HNE activity rapidly reappeared at the surface of HNE-depleted cells when they were triggered with the calcium ionophore A23187, and this activity was fully inhibited by stoichiometric amounts of alpha1-Pi. HNE was not released from the cell surface by oxidized, inactive alpha1-Pi, showing that active inhibitor is required to interact with active protease from the cell surface. We conclude that HNE activity at the surface of human neutrophils is fully controlled by alpha1-Pi when the cells are in suspension. Pericellular proteolysis could be limited to zones of contact between neutrophils and subjacent protease substrates where natural inhibitors cannot penetrate.     

Angiogenic activity of bFGF and VEGF suppressed by proteolytic cleavage by neutrophil elastase

Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation.     

Blockade of neutrophil elastase attenuates severe liver injury in hepatitis B transgenic mice

Serine proteinases produced by polymorphonuclear neutrophils play important roles in neutrophil-mediated tissue injury at inflammatory sites. Although neutrophil recruitment to the liver has been shown to be involved in the exacerbation of liver inflammation, the function of neutrophil elastase (NE) in liver injury remains unclear. Here, we found that administration of an NE inhibitor (NEI) reduced serum alanine aminotransferase (sALT) activity and inflammatory cell infiltration into the liver from 8 to 24 h after injection of antigen-specific cytotoxic T lymphocytes (CTLs) into hepatitis B virus transgenic mice. Furthermore, the NEI treatment reduced the expressions of inflammatory cytokines and chemokines in the liver and tumor necrosis factor alpha production by macrophages. In addition, the NEI treatment suppressed the mRNA expressions of CC chemokine ligand 3 (CCL-3), CCL-4, and macrophage inflammatory protein 2 (MIP-2) in neutrophils in the liver at 8 h after the CTL injection. In support of these results, we confirmed that administration of anti-CCL-3, anti-CCL-4, and anti-MIP-2 monoclonal antibodies suppressed sALT activity and leukocyte migration into the liver. In conclusion, the present results suggest that NE contributes to the early step of the inflammatory cascade in acute viral hepatitis and that NEIs may have potential as therapeutic drugs against acute severe viral hepatitis.     

Proteinase-3 as the major autoantigen of c-ANCA is strongly expressed in lung tissue of patients with Wegener's granulomatosis

Proteinase-3 (PR-3) is a neutral serine proteinase present in azurophil granules of human polymorphonuclear leukocytes and serves as the major target antigen of antineutrophil cytoplasmic antibodies with a cytoplasmic staining pattern (c-ANCA) in Wegener's granulomatosis (WG). The WG disease appears as severe vasculitis in different organs (e.g. kidney, nose and lung). Little is known about the expression and distribution of PR-3 in the lung. We found that PR-3 is expressed in normal lung tissue and is upregulated in lung tissue of patients with WG. Interestingly, the parenchymal cells (pneumocytes type I and II) and macrophages, and not the neutrophils, express PR-3 most strongly and may contribute to lung damage in patients with WG via direct interaction with antineutrophil cytoplasmic antobodies (ANCA). These findings suggest that the PR-3 expression in parenchymal cells of lung tissue could be at least one missing link in the etiopathogenesis of pulmonary pathology in ANCA-associated disease.     

Alpha-1-antichymotrypsin inhibits the NADPH oxidase-enzyme complex in phorbol ester-stimulated neutrophil membranes.

The generation of superoxide anion and release of granule contents are essential to the bactericidal function of neutrophils, but may also contribute to host tissue damage during inflammation. In previous studies (J. Immunol. 146:2388), we have demonstrated that the acute phase reactant alpha-1-antichymotrypsin (ACT), a potent inhibitor of the serine protease cathepsin G, also suppresses superoxide anion generation. The inhibitory effect of ACT was not directly linked to its antiproteolytic activity and may reflect interaction at a site other than its reactive loop. To further characterize the mechanism of inhibition, we investigated the direct effects of ACT on the NADPH oxidase enzyme complex and the signaling pathways that regulate motivation of the respiratory burst. We present evidence that ACT does not intefer with agonist-stimulated calcium mobilization or translocation and activity of protein kinase C. ACT was an effective inhibitor of superoxide anion generation in membrane preparations isolated from PMA-activated cells. These results support the notion that ACT is acting on a component of the active assembled NADPH oxidase complex. Thus, ACT may have an important role in regulation of specific aspects of the inflammatory processes and the modulation of toxic oxygen-based host tissue damage.     

Proteinase-3 as the major autoantigen of c-ANCA is strongly expressed in lung tissue of patients with Wegener's granulomatosis

Proteinase-3 (PR-3) is a neutral serine proteinase present in azurophil granules of human polymorphonuclear leukocytes and serves as the major target antigen of antineutrophil cytoplasmic antibodies with a cytoplasmic staining pattern (c-ANCA) in Wegener's granulomatosis (WG). The WG disease appears as severe vasculitis in different organs (e.g. kidney, nose and lung). Little is known about the expression and distribution of PR-3 in the lung. We found that PR-3 is expressed in normal lung tissue and is upregulated in lung tissue of patients with WG. Interestingly, the parenchymal cells (pneumocytes type I and II) and macrophages, and not the neutrophils, express PR-3 most strongly and may contribute to lung damage in patients with WG via direct interaction with antineutrophil cytoplasmic antobodies (ANCA). These findings suggest that the PR-3 expression in parenchymal cells of lung tissue could be at least one missing link in the etiopathogenesis of pulmonary pathology in ANCA-associated disease.     

Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Apoptosis of human neutrophils induced by protein phosphatase 1/2A inhibition is caspase-independent and serine protease-dependent

Protein phosphatase (PP) activity is associated with the regulation of apoptosis in neutrophils. However, the underlying regulatory mechanism(s) in apoptosis remain unclear. The type of cell death induced by okadaic acid (OA), the inhibitor of PP1 and PP2A, is characterized by apoptotic morphological changes of the cells and annexin V-positive staining without DNA fragmentation. The apoptotic effects of OA and calyculin A on neutrophils were observed at concentrations ranging from 50 to 200 nM, or 10 to 50 nM, respectively. Cyclosporine A (a PP2B specific inhibitor), however, did not exhibit any pro-apoptotic effects. OA and calyculin A, but not cyclosporine A, exhibited significant effects on protein levels and on the electrophoretic mobility of Mcl-1. zVAD-fmk, a pancaspase inhibitor, failed to inhibit the effect of OA on the caspase-3 activity, procaspase-3 processing, and the apoptotic rate of neutrophils. However, 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), a general serine protease inhibitor, significantly abrogated the OA-induced mobility shift in procaspase-3, caspase-3 activation, and the apoptotic morphological changes in neutrophils. Moreover, OA enhanced the serine protease activity of the neutrophils. The addition of the proteinase-3 protein increased the rate of neutrophil apoptosis, which was also blocked by AEBSF but not by zVAD-fmk. These results suggest that OA induces procaspase-3 processing but that OA-induced apoptosis is caspase-independent and serine protease-dependent.     

Altered protease and antiprotease balance during a COPD exacerbation contributes to mucus obstruction

Background

Proteases have been shown to degrade airway mucin proteins and to damage the epithelium impairing mucociliary clearance. There are increased proteases in the COPD airway but changes in protease-antiprotease balance and mucin degradation have not been investigated during the course of a COPD exacerbation. We hypothesized that increased protease levels would lead to mucin degradation in acute COPD exacerbations.

Methods

We measured neutrophil elastase (NE) and alpha 1 protease inhibitor (A1-PI) levels using immunoblotting, and conducted protease inhibitor studies, zymograms, elastin substrate assays and cigarette smoke condensate experiments to evaluate the stability of the gel-forming mucins, MUC5AC and MUC5B, before and 5–6 weeks after an acute pulmonary exacerbation of COPD (n = 9 subjects).

Results

Unexpectedly, mucin concentration and mucin stability were highest at the start of the exacerbation and restored to baseline after 6 weeks. Consistent with these data, immunoblots and zymograms confirmed decreased NE concentration and activity and increased A1-PI at the start of the exacerbation. After recovery there was an increase in NE activity and a decrease in A1-PI levels. In vitro, protease inhibitor studies demonstrated that serine proteases played a key role in mucin degradation. Mucin stability was further enhanced upon treating with cigarette smoke condensate (CSC).

Conclusion

There appears to be rapid consumption of secreted proteases due to an increase in antiproteases, at the start of a COPD exacerbation. This leads to increased mucin gel stability which may be important in trapping and clearing infectious and inflammatory mediators, but this may also contribute acutely to mucus retention.     

Granulocyte and plasma cytokine activity in acute cadmium intoxication in rats.

Changes in the number and ex vivo function of peripheral blood neutrophils were investigated following intraperitoneal administration of cadmium-chloride in rats. Besides a dose-dependent increase in the number of peripheral blood neutrophils, changes were found in the functional state of isolated polymorphonuclear leukocytes (PMNs). Increased spontaneous adhesion and activation, and TNF activity in a conditioned medium were observed in cultures of granulocytes in comparison to granulocytes from control (saline-treated) animals. Increased levels of plasma activity of inflammatory cytokines, tumor necrosis factor (TNF) and interleukin-6 (IL-6) were noted following cadmium administration. Cytological signs of pulmonary inflammation were revealed histologically and the majority of neutrophils recovered from the lungs by enzyme digestion exhibited a capacity of nitroblue tetrazolium (NBT) reduction. Our data demonstrate that acute cadmium intoxication leads to a systemic inflammatory response characterized by numerical and functional changes in the granulocyte compartment and to increased levels of inflammation-related cytokine activity in the circulation. Correlations between the increased number of peripheral blood neutrophils and IL-6 plasma activity (r=0.776, p<0.00001) and the number of neutrophils recovered from the lung tissue (r=0.893, p<0.00001) suggested that systemic cadmium-induced inflammation might be involved in the pulmonary toxicity of cadmium.