Generation and functional significance of CXC chemokines for neutrophil-induced liver injury during endotoxemia

The hypothesis that the neutrophil chemoattractant CXC chemokines KC and macrophage inflammatory protein-2 (MIP-2) are involved in neutrophil transmigration and liver injury was tested in C3Heb/FeJ mice treated with galactosamine (Gal, 700 mg/kg), endotoxin (ET, 100 microg/kg), or Gal + ET (Gal/ET). Hepatic KC and MIP-2 mRNA levels and plasma CXC chemokine concentrations were dramatically increased 1.5 h after Gal/ET or ET alone and gradually declined up to 7 h. Murine recombinant cytokines (TNF-alpha, IL-1 alpha, and IL-1 beta), but not Gal/ET, induced CXC chemokine formation in the ET-resistant C3H/HeJ strain. To assess the functional importance of KC and MIP-2, C3Heb/FeJ mice were treated with Gal/ET and control IgG or a combination of anti-KC and anti-MIP-2 antibodies. Anti-CXC chemokine antibodies did not attenuate hepatocellular apoptosis, sinusoidal neutrophil sequestration and extravasation, or liver injury at 7 h. Furthermore, there was no difference in liver injury between BALB/cJ wild-type and CXC receptor-2 gene knockout (CXCR2-/-) mice treated with Gal/ET. The higher neutrophil count in livers of CXCR2-/- than in wild-type mice after Gal/ET was caused by the elevated number of neutrophils located in sinusoids of untreated CXCR2-/- animals. The pancaspase inhibitor Z-Val-Ala-Asp-fluoromethylketone eliminated Gal/ET-induced apoptosis and neutrophil extravasation and injury but not CXC chemokine formation. Thus Gal/ET induced massive, cytokine-dependent CXC chemokine formation in the liver. However, neutrophil extravasation and injury occurred in response to apoptotic cell injury at 6-7 h and was independent of CXC chemokine formation.     

NADPH oxidase-derived oxidant stress is critical for neutrophil cytotoxicity during endotoxemia

Neutrophils can cause liver injury during endotoxemia through generation of reactive oxygen species. However, the enzymatic source of the oxidant stress and the nature of the oxidants generated remain unclear. Therefore, we investigated the involvement of NADPH oxidase in the pathophysiology by using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) in the galactosamine/endotoxin (700 mg/kg Gal:100 microg/kg ET) model of liver injury. In addition, we measured chlorotyrosine as indicator for hypochlorous acid formation by myeloperoxidase. Gal/ET treatment of male C3HeB/FeJ mice resulted in sinusoidal neutrophil accumulation and parenchymal cell apoptosis (14 +/- 3% of cells) at 6 h. At 7 h, 35% of neutrophils had transmigrated. The number of apoptotic cells increased to 25 +/- 2%, and the overall number of dead cells was 48 +/- 3%; many of them showed the characteristic morphology of necrosis. Hepatocytes, which colocalized with extravasated neutrophils, stained positive for chlorotyrosine and 4-hydroxynonenal (4-HNE) protein adducts. In contrast, animals pretreated with DPI (2.5 mg/kg) were protected against liver injury at 7 h (necrosis = 20 +/- 2%). These livers showed little chlorotyrosine or 4-HNE staining, but apoptosis and neutrophil accumulation and extravasation remained unaffected. However, DPI-treated animals showed serious liver injury at 9 h due to sustained apoptosis. The results indicate that NADPH oxidase is responsible for the neutrophil-derived oxidant stress, which includes formation of hypochlorous acid by myeloperoxidase. Thus NADPH oxidase could be a promising therapeutic target to prevent neutrophil-mediated liver injury. However, the long-term benefit of this approach needs to be investigated in models relevant for human liver disease.     

Generation of hypochlorite-modified proteins by neutrophils during ischemia-reperfusion injury in rat liver: attenuation by ischemic preconditioning

Although it is well documented that neutrophils are critical for the delayed phase of hepatic ischemia-reperfusion injury, there is no direct evidence for a specific neutrophil-derived oxidant stress in vivo. Therefore, we used a model of 60 min of partial hepatic ischemia and 0-24 h of reperfusion to investigate neutrophil accumulation and to analyze biomarkers for a general oxidant stress [glutathione disulfide (GSSG) and malondialdehyde (MDA)] and for a neutrophil-specific oxidant stress [hypochlorite (HOCl)-modified epitopes] in rats. Plasma alanine transaminase activities and histology showed progressively increasing liver injury during reperfusion, when hepatic GSSG and soluble MDA levels were elevated. At that time, few neutrophils were present in sinusoids. However, the number of hepatocytes positively stained for HOCl-modified epitopes increased from 6 to 24 h of reperfusion, which correlated with the bulk of hepatic neutrophil accumulation and extravasation into the parenchyma. Consistent with a higher oxidant stress at later times, hepatic GSSG and protein-bound MDA levels further increased. Treatment with the NADPH oxidase inhibitor diphenyleneiodonium chloride attenuated postischemic oxidant stress (GSSG, protein-bound MDA, and hepatocytes positively stained for HOCl-modified epitopes) and liver injury at 24 h of reperfusion. Ischemic preconditioning suppressed all oxidant stress biomarkers, liver injury, and extravasation of neutrophils. In conclusion, extravasated neutrophils generate HOCl, which diffuses into hepatocytes and causes oxidative modifications of intracellular proteins during the neutrophil-mediated reperfusion injury phase. Ischemic preconditioning is an effective intervention for reduction of the overall inflammatory response and, in particular, for limitation of the cytotoxic activity of neutrophils during the later reperfusion period.     

Embolization by sinusoidal lining cells obstructs the microcirculation in rat sinusoidal obstruction syndrome

Mechanisms leading to the obstruction of the microcirculation in sinusoidal obstruction syndrome (SOS) have been unclear. Because this occurs at the onset of disease, this is a potential key target for therapeutic intervention. Rats were treated with monocrotaline with or without continuous intraportal infusion of glutathione and were studied at 0.5, 1, 2, 4, 6, and 10 days after monocrotaline treatment with the use of in vivo microscopy and transmission electron microscopy. Sinusoidal perfusion decreased from days 1 through 10 with a nadir on day 4. At 12 h, numerous swollen sinusoidal endothelial cells (SECs) were observed. Subsequently, red blood cells penetrated into the space of Disse through gaps between and through swollen SEC and dissected the sinusoidal lining away from the parenchymal cells. Sinusoidal blood flow was obstructed by an embolism of aggregates of sinusoidal lining cells, red blood cells, and adherent monocytes. All changes were prevented by glutathione infusion, notably the initial swelling of SEC. SOS is initiated by changes in SEC. Microcirculatory obstruction is due to dissection of the sinusoidal lining, followed by embolization of the sinusoid by sinusoidal lining cells, compounded by aggregates of monocytes adherent in the sinusoids. Glutathione prevents SOS by preserving an intact sinusoidal barrier.     

Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN

High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17-18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.     

Time-dependent inhibition of immune complex-induced lung injury by catalase: relationship to alterations in macrophage and neutrophil matrix metalloproteinase elaboration

Rats were subjected to acute lung injury by the intra-alveolar formation of IgG immune complexes of bovine serum albumin (BSA) and anti-BSA. In this model of injury, complement activation occurs and large numbers of neutrophils invade the interstitium and alveolar space. In the present study, animals were treated with intratracheal catalase concomitantly with anti-BSA or after a lag period of 5-120 min. Catalase treatment at time-zero or at 5 min post injury failed to prevent lung injury as indicated by permeability change, histological features, and neutrophil influx. However, treatment after a delay of 15-30 min (but not 120 min) afforded substantial protection. Consistent with past findings [19], lung injury was accompanied by an accumulation of matrix metalloproteinase 9 (MMP-9) in bronchoalveolar lavage (BAL) fluid. There was a strong correlation between inhibition of injury and reduction in MMP-9 levels. In vitro studies conducted in parallel revealed that unstimulated alveolar macrophages did not produce measurable MMP-9, while there was a large induction following exposure to the same immune complexes that initiated injury in vivo. MMP-2 was also slightly upregulated under the same conditions. Concomitant treatment with catalase greatly inhibited MMP-9 production by macrophages in response to immune complexes, but this treatment had little effect on basal production of either MMP-9 or MMP-2 by macrophage. The same concentration of catalase that suppressed MMP-9 elaboration also inhibited the production of tumor necrosis factor alpha. In contrast, when neutrophils were treated with catalase and then exposed to immune complexes, the antioxidant failed to prevent the release of either MMP-2 or MMP-9. Taken together, these findings demonstrate that antioxidant treatment interferes with elaboration of MMPs by alveolar macrophages. Protection against lung injury is correlated with reduction in MMP levels in the BAL fluid.     

A neutrophil elastase inhibitor (ONO-5046) reduces neurologic damage after spinal cord injury in rats

In view of a cytoprotective effect of elastase inhibitor on chemokine-mediated tissue injury, we examined the neuroprotective effect of ONO-5046, a specific inhibitor of neutrophil elastase, in rats with spinal cord injury. Standardized spinal cord compression markedly increased cytokine-induced neutrophil chemo-attractant (CINC)-1 mRNA and protein. Their increases correlated with neurologic severity of injured rats. Immunohistochemically, CINC-1 protein was detected sequentially in vascular endothelial cells at 4 h, in perivascular neutrophils at 8 h, and in neutrophils infiltrating into cord substance at 12 h. Pretreatment with ONO-5046 (50 mg/kg) markedly ameliorated motor disturbance in injured rats, and reduced CINC-1 protein and mRNA expression. ONO-5046 also significantly reduced the increase of neutrophil accumulation or infiltration estimated by myeloperoxidase activity, and the extent of vascular permeability by Evans blue extravasation in the injured cord segment in comparison to control animals receiving vehicle. These results suggest that CINC-1 contributed to inflammation in rat spinal cord injury and ONO-5046 attenuated neurologic damage partly by blocking CINC-1 production of the chemoattractant, preventing neutrophil activation and vascular endothelial cell injury.     

Effects of CXC chemokines on neutrophil activation and sequestration in hepatic vasculature

The initiating step of neutrophil-induced cytotoxicity in the liver is the recruitment of these phagocytes into sinusoids. The aim of our study was to compare the efficacy of systemic exposure with individual inflammatory mediators on neutrophil activation and sequestration in the hepatic vasculature of C3Heb/FeJ mice as assessed by flow cytometry and histochemistry, respectively. The CXC chemokine macrophage inflammatory protein-2 (MIP-2; 20 microg/kg) induced a time-dependent upregulation of Mac-1 (318% at 4 h) and shedding of L-selectin (41% at 4 h). MIP-2 treatment caused a temporary increase of sinusoidal neutrophil accumulation at 0.5 h [97 +/- 6 polymorphonuclear leukocytes (PMN)/50 high-power fields (HPF)], which declined to baseline (8 +/- 2) at 4 h. The CXC chemokine KC was largely ineffective in activating neutrophils or recruiting them into the liver. Cytokines (tumor necrosis factor-alpha and interleukin-1alpha) and cobra venom factor substantially increased Mac-1 expression and L-selectin shedding on neutrophils and caused stable sinusoidal neutrophil accumulation (170-220 PMN/50 HPF). Only cytokines induced venular neutrophil margination. Thus CXC chemokines in circulation are less effective than cytokines or complement in activation of neutrophils and their recruitment into the hepatic vasculature in vivo.     

Matrix Metalloproteinase-2 (MMP-2) Gene Deletion Enhances MMP-9 Activity,Impairs PARP-1 Degradation,and Exacerbates Hepatic Ischemia and Reperfusion Injury in Mice

Hepatic ischemia and reperfusion injury (IRI) is an inflammatory condition and a significant cause of morbidity and mortality after surgery. Matrix metalloproteinases (MMPs) have been widely implicated in the pathogenesis of inflammatory diseases. Among the different MMPs, gelatinases (MMP-2 and MMP-9) are within the most prominent MMPs detected during liver IRI. While the role of MMP-9 in liver damage has been fairly documented, direct evidence of the role for MMP-2 activity in hepatic IRI remains to be established. Due to the lack of suitable inhibitors to target individual MMPs in vivo, gene manipulation is as an essential tool to assess MMP direct contribution to liver injury. Hence, we used MMP-2^-/- deficient mice and MMP-2^+/+ wild-type littermates to examine the function of MMP-2 activity in hepatic IRI. MMP-2 expression was detected along the sinusoids of wild-type livers before and after surgery and in a small population of leukocytes post-IRI. Compared to MMP-2^+/+ mice, MMP-2 null (MMP-2^-/-) mice showed exacerbated liver damage at 6, 24, and 48 hours post-reperfusion, which was fatal in some cases. MMP-2 deficiency resulted in upregulation of MMP-9 activity, spontaneous leukocyte infiltration in naïve livers, and amplified MMP-9-dependent transmigration of leukocytes in vitro and after hepatic IRI. Moreover, complete loss of MMP-2 activity impaired the degradation of poly (ADP-ribose) polymerase (PARP-1) in extensively damaged livers post-reperfusion. However, the administration of a PARP-1 inhibitor to MMP-2 null mice restored liver preservation to almost comparable levels of MMP-2^+/+ mice post-IRI. Deficient PARP-1 degradation in MMP-2-null sinusoidal endothelial cells correlated with their increased cytotoxicity, evaluated by the measurement of LDH efflux in the medium. In conclusion, our results show for the first time that MMP-2 gene deletion exacerbates liver IRI. Moreover, they offer new insights into the MMP-2 modulation of inflammatory responses, which could be relevant for the design of new pharmacological MMP-targeted agents to treat hepatic IRI.     

ET(A)-receptor blockade prevents matrix metalloproteinase activation late postmyocardial infarction in the rat

Endothelin (ET) A (ET(A)) receptors activate matrix metalloproteinases (MMP). Since endothelin-1 (ET) is increased in myocardium late postmyocardial infarction (MI), we hypothesized that stimulation of ET(A) receptors contributes to activation of myocardial MMPs late post-MI. Three days post-MI, rats were randomized to treatment with the ET(A)-selective receptor antagonist sitaxsentan (n = 12) or a control group (n = 12). Six weeks later, there were rightward shifts of the left ventricular (LV) end-diastolic and end-systolic pressure-volume relationships, as measured ex vivo by the isovolumic Langendorff technique. Both shifts were markedly attenuated by sitaxsentan. In LV myocardium remote from the infarct, the activities of MMP-1, MMP-2, and MMP-9 were increased in the post-MI group, and the increases were prevented by sitaxsentan treatment. Expression of tissue inhibitor of MMP-1 was decreased post-MI, and the decrease was prevented by sitaxsentan treatment. Chronic post-MI remodeling is associated with activation of MMPs in myocardium remote from the infarct. Inhibition of ET(A) receptors prevents MMP activation and LV dilation, suggesting that ET, acting via the ET(A) receptor, contributes to chronic post-MI remodeling by its effects on MMP activity.     

The profile of MMP and TIMP in injured rat ACL

Human anterior cruciate ligament (ACL) has poor healing ability after injury. The devastating effects of matrix metalloproteinases (MMPs) excess expression are regarded as the main reason. Tissue inhibitor metalloproteinases (TIMPs) may be independent of MMPs inhibition. In this paper, a rat ACL rotating injury apparatus was designed to produce ACL injury. After inducing injury, joint fluids and ACL tissue total proteins were immediately extracted. In addition, ACL tissue was isolated in a culture plate with 1%FBS medium for the ex vivo study. We found MMP-2 in joint fluids increased significantly by 4 folds after ACL injury as a function of time. Ex vivo study showed MMP-2 in the medium and ACL cultured tissue increased significantly respectively to 3 folds and to 6 folds. The joint fluids global MMP increased to 3.5 folds with non-treatment and APMA-treatment in day three. On the gene expression level, the changes in MMP-1 and CD147 have the similar tendency. The ratio of MMP-1/TIMP-1 increased with time after ACL injury. We conclude that MMP-2 increased significantly in the early phase in the joint cavity after ACL injury. The ex vivo study demonstrated the same tendency. Generic MMP Activity Assay (global MMP assay) an dzymography also showed significant increase in MMP activity in joint fluids. These results showed ACL having poor healing ability after injury may not be only due to ACL release of large quantities of MMPs. The important factor may be the alterations in the whole joint cavity's internal environment.     

Inhibition of matrix metalloproteinases blocks lethal hepatitis and apoptosis induced by tumor necrosis factor and allows safe antitumor therapy.

Acute and fulminant liver failure induced by viral hepatitis, alcohol or other hepatotoxic drugs, are associated with tumor necrosis factor (TNF) production. In a mouse model of lethal hepatitis induced by TNF, apoptosis and necrosis of hepatocytes, but also lethality, hypothermia and influx of leukocytes into the liver, are prevented by a broad-spectrum matrix metalloproteinase (MMP) inhibitor, BB-94. Mice deficient in MMP-2, MMP-3 or MMP-9 had lower levels of apoptosis and necrosis of hepatocytes, and better survival. We found induction of MMP-9 activity and fibronectin degradation. Our findings suggest that several MMPs play a critical role in acute, fulminant hepatitis by degrading the extracellular matrix and allowing massive leukocyte influx in the liver. BB-94 also prevented lethality in TNF/interferon-gamma therapy in tumor-bearing mice. A broad-spectrum MMP inhibitor may be potentially useful for the treatment of patients with acute and perhaps chronic liver failure, and in cancer therapies using inflammatory cytokines.     

Matrix metalloproteinases inhibition provides neuroprotection against hypoxia-ischemia in the developing brain

The present study was designed to investigate the role of matrix metalloproteinases (MMPs) in the immature brain and the long term effects of early MMPs inhibition after hypoxic-ischemic (HI) injury. HI was induced by unilateral ligation of the right carotid artery followed by hypoxia (8% O₂ for 2 h) in P7 rat pups. GM6001, a broad spectrum MMPs inhibitor, was injected (50 mg/kg or 100 mg/kg) intraperitoneally at 2 h and 24 h after HI injury. Blood-brain barrier (BBB) integrity, brain edema, MMP-2/-9 activity, TIMP-1/-2 and tight junction protein (TJP) level were evaluated using IgG staining, Evan's blue extravasation, brain water content, zymography and western blot. Doxycycline, another MMPs inhibitor, was injected (10 mg/kg or 30 mg/kg) intraperitoneally at 2 h after HI, then BBB integrity and brain edema were measured at 48 h post-HI using brain water content measurement and IgG staining. The long-term effects of early MMPs inhibition (GM6001, 100 mg/kg) were evaluated by neurobehavioral tests, body weight, and brain atrophy measurement. GM6001 attenuated brain edema and BBB disruption at the dosage of 100 mg/kg. MMP-2 activity increased at 24 h and peaked at 48 h after HI, whereas MMP-9 activity peaked at 24 h and tapered by 48 h after HI. MMP-9/-2 activities were significantly attenuated by GM6001 at 24 h and 48 h after HI. The degradation of TJPs (ZO-1 and occludin) at 48 h after HI was reversed by GM6001 treatment. Early MMPs inhibition had long-term effects that attenuated ipsilateral brain tissue loss, and improved neurobehavioral outcomes after HI. These results suggest that early MMPs inhibition with a broad-spectrum inhibitor provides both acute and long-term neuroprotection in the developing brain by reducing TJPs degradation, preserving BBB integrity, and ameliorating brain edema after neonatal HI injury.     

Reduction of myocardial infarct size by doxycycline: A role for plasmin inhibition

Myocardial ischemia-reperfusion (I/R) is associated with the activation of matrix metalloproteinases (MMPs) and serine proteases. We hypothesized that activation of MMPs and the serine protease plasmin contribute to early cardiac myocyte death following I/R and that broad-spectrum protease inhibition with doxycycline (DOX) preserves myocyte viability. Rats treated daily with or without DOX beginning 48 h prior to experimentation were subjected to 30 min of coronary occlusion and 2 days of reperfusion. DOX pre-treatment reduced infarct size by 37%. DOX attenuated increases in MMP-9 and plasmin levels as determined by gelatin zymography and immunoblot, respectively. Neutrophil extravasation was unaltered by DOX as assessed by myeloperoxidase (MPO) activity. To examine the contribution of MMP-9 and plasmin to myocyte injury, cultures of neonatal rat ventricular myocytes (NRVMs) were treated for 48 h with 83 kDa MMP-9 or plasminogen in the presence or absence of DOX. MMP-9 treatment did not affect myocyte viability. Plasminogen treatment led to increased plasmin activity, resulting in loss of ₁-integrin, NRVM detachment and apoptosis. DOX co-treatment inhibited plasmin activity and preserved NRVM attachment, whereas co-treatment with the broad-spectrum MMP inhibitor GM6001 had no effect. These results indicate that plasmin causes disruption of myocyte attachment and viability independently of MMP activation in vitro and that inhibition of plasmin by DOX may reduce I/R-induced myocyte death in vivo through the inhibition of plasmin. (Mol Cell Biochem 270: 1–11, 2005)     

Polymorphonuclear leukocytes promote neurotoxicity through release of matrix metalloproteinases, reactive oxygen species, and TNF-alpha

As the first immune cells to infiltrate the nervous system after traumatic PNS and CNS injury, neutrophils (polymorphonuclear leukocytes, PMNs) may promote injury by releasing toxic soluble factors that may affect neuronal survival. Direct neurotoxicity of matrix metalloproteinases (MMPs), reactive oxygen species (ROS), and cytokines released by PMNs was investigated by culturing dorsal root ganglion (DRG) cells with PMN-conditioned media containing MMP inhibitor (GM6001), ROS scavengers, or tumor necrosis factor alphaR (TNF-alphaR) neutralizing antibody. Although DRGs exposed to PMN-conditioned media had 53% fewer surviving neurons than controls, neuronal cell loss was prevented by GM6001 (20 micromol/L), catalase (1000 U/mL), or TNF-alphaR neutralizing antibody (1.5 microg/mL), elevating survival to 77%, 94%, and 95%, respectively. In accordance with protection by GM6001, conditioned media collected from MMP-9 null PMNs was less neurotoxic than that collected from wild-type PMNs. Additionally, MMP inhibition reduced PMN-derived ROS; removal of ROS reduced PMN-derived MMP-9 activity; and TNF-alpha inhibition reduced both PMN-derived MMP-9 activity and ROS in PMN cultures. Our data provide the first direct evidence that PMN-driven neurotoxicity is dependent on MMPs, ROS, and TNF-alpha, and that these factors may regulate PMN release of these soluble factors or interact with one another to mediate PMN-driven neurotoxicity.     

Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug

Tumor necrosis factor (TNF) prodrugs are fusion proteins comprised of an N-terminal single-chain antibody variable fragment (scFv) targeting a TNF effector and a C-terminal TNF receptor (TNFR)1-derived inhibitor module. Introduction of matrix metalloproteinase (MMP)-2 recognition motifs between TNF and the TNFR1 fragment allowed activation by recombinant MMP-2 and MMP-expressing HT1080 cells. Processing by endogeneous MMPs required specific membrane binding of the TNF prodrug via the targeting scFv, ensuring strictly antigen-dependent activation. Interestingly, TNF bioactivity of the processed prodrug was approximately 1000-fold higher upon scFv-mediated targeting, and signaled juxtatropic cell death also to antigen-negative cells. Microscopical analyses of TNFR2 clustering and TNF receptor-associated factor 2 recruitment at contact sites to adjacent cells revealed the formation of stable TNFR complexes by target-bound, processed prodrug, resembling the increased signal capacity of natural, membrane-expressed TNF. MMP-2-sensitive TNF prodrugs represent novel cytokine-based reagents for targeted cancer therapy, which should be exploitable for MMP-overexpressing tumors.     

Matrix metalloproteinase 9 activity enhances host susceptibility to pulmonary infection with type A and B strains of Francisella tularensis

A striking feature of pulmonary infection with the Gram-negative intracellular bacterium Francisella tularensis, a category A biological threat agent, is an intense accumulation of inflammatory cells, particularly neutrophils and macrophages, at sites of bacterial replication. Given the essential role played by host matrix metalloproteinases (MMPs) in modulating leukocyte recruitment and the potentially indiscriminate destructive capacity of these cells, we investigated whether MMP-9, an important member of this protease family released by neutrophils and activated macrophages, plays a role in the pathogenesis of respiratory tularemia. We found that F. tularensis induced expression of MMP-9 in FVB/NJ mice and that the action of this protease is associated with higher bacterial burdens in pulmonary and extrapulmonary tissues, development of more extensive histopathology predominated by neutrophils, and increased morbidity and mortality compared with mice lacking MMP-9 (MMP-9(-/-)). Moreover, MMP-9(-/-) mice were able to resolve infection with either the virulence-attenuated type B (live vaccine strain) or the highly virulent type A (SchuS4) strain of F. tularensis. Disease resolution was accompanied by diminished leukocyte recruitment and reductions in both bacterial burden and proinflammatory cytokine production. Notably, neutrophilic infiltrates were significantly reduced in MMP-9(-/-) mice, owing perhaps to limited release of Pro-Gly-Pro, a potent neutrophil chemotactic tripeptide released from extracellular matrix through the action of MMP-9. Collectively, these results suggest that MMP-9 activity plays a central role in modulating the clinical course and severity of respiratory tularemia and identifies MMPs as novel targets for therapeutic intervention as a means of modulating neutrophil recruitment.     

Matrix metalloproteinase secretion by gastric epithelial cells is regulated by E prostaglandins and MAPKs

Because matrix metalloproteinases (MMPs) play roles in inflammatory tissue injury, we asked whether MMP secretion by gastric epithelial cells may contribute to gastric injury in response to signals involved in Helicobacter pylori-induced inflammation and/or cyclooxygenase inhibition. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and epidermal growth factor (EGF) stimulated gastric cell MMP-1 secretion, indicating that MMP-1 secretion occurs in inflammatory as well as non-inflammatory situations. MMP-1 secretion required activation of the MAPK Erk and subsequent protein synthesis but was down-regulated by the alternate MAPK, p38. In contrast, secretion of MMP-13 was stimulated by TNF-alpha/IL-1beta but not EGF and was Erk-independent and mediated by p38. MMP-13 secretion was more rapid (peak, 6 h) than MMP-1 (peak > or =30 h) and only partly depended on protein synthesis, suggesting initial release of a pre-existing MMP-13 pool. Therefore, MMP-1 and MMP-13 secretion are differentially regulated by MAPKs. MMP-1 secretion was regulated by E prostaglandins (PGEs) in an Erk-dependent manner. PGEs enhanced Erk activation and MMP-1 secretion in response to EGF but inhibited Erk and MMP-1 when TNF-alpha and IL-1beta were the stimuli, indicating that the effects of PGEs on gastric cell responses are context-dependent. These data show that secretion of MMPs is differentially regulated by MAPKs and suggest mechanisms through which H. pylori infection and/or cyclooxygenase inhibition may induce epithelial cell signaling to contribute to gastric ulcerogenesis.     

Transient expression of laminin alpha1 chain in regenerating murine liver: restricted localization of laminin chains and nidogen-1

Most interstitia between epithelial and endothelial cells contain basal laminae (BLs), as defined by electron microscopy. However, in liver, the sinusoidal interstitium (called space of Disse) between hepatocytes and sinusoidal endothelial cells (SECs) lacks BLs. Because laminins are major components of BLs throughout the body, whether laminins exist in sinusoids has been a controversial issue. Despite recent advances, the distribution and expression of laminin chains have not been well defined in mammalian liver. Here, using a panel of antibodies, we examined laminins in normal and regenerating mouse livers. Of alpha chains, alpha5 was widely observed in all BLs except for sinusoids, while the other alpha chains were variously expressed in Glisson's sheath and central veins. Laminin gamma1 was also distributed to all BLs except for sinusoids. Although the beta2 chain was observed in all BLs and sinusoids, the expression of beta1 chain was restricted to Glisson's sheath. Detailed analysis of regenerating liver revealed that alpha1 and gamma1 chains appeared in sinusoids and were produced by stellate cells. The staining of alpha1 and gamma1 chains reached its maximum intensity at 6 days after two-thirds partial hepatectomy (PHx). Moreover, in vitro studies showed that alpha1-containing laminin promoted spreading of sinusoidal endothelial cells (SECs) isolated from normal liver, but not other hepatic cells. In addition, SECs isolated from regenerating liver elongated pseudopodia on alpha1-containing laminin more so than did cells from normal liver. The transient expression of laminin alpha1 may promote formation of sinusoids after PHx.     

Cigarette smoke-induced lung emphysema in mice is associated with prolyl endopeptidase, an enzyme involved in collagen breakdown

There is increasing evidence that the neutrophil chemoattractant proline-glycine-proline (PGP), derived from the breakdown of the extracellular matrix, plays an important role in neutrophil recruitment to the lung. PGP formation is a multistep process involving neutrophils, metalloproteinases (MMPs), and prolyl endopeptidase (PE). This cascade of events is now investigated in the development of lung emphysema. A/J mice were whole body exposed to cigarette smoke for 20 wk. After 20 wk or 8 wk after smoking cessation, animals were killed, and bronchoalveolar lavage fluid and lung tissue were collected to analyze the neutrophilic airway inflammation, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels. Lung tissue degradation was assessed by measuring the mean linear intercept. Additionally, we investigated the effect of the peptide L-arginine-threonine-arginine (RTR), which binds to PGP sequences, on the smoke-induced neutrophil influx in the lung after 5 days of smoke exposure. Neutrophilic airway inflammation was induced by cigarette smoke exposure. MMP-8 and MMP-9 levels, PE activity, and PGP levels were elevated in the lungs of cigarette smoke-exposed mice. PE was highly expressed in epithelial and inflammatory cells (macrophages and neutrophils) in lung tissue of cigarette smoke-exposed mice. After smoking cessation, the neutrophil influx, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels were decreased or reduced to normal levels. Moreover, RTR inhibited the smoke-induced neutrophil influx in the lung after 5 days' smoke exposure. In the present murine model of cigarette smoke-induced lung emphysema, it is demonstrated for the first time that all relevant components (neutrophils, MMP-8, MMP-9, PE) involved in PGP formation from collagen are upregulated in the airways. Together with MMPs, PE may play an important role in the formation of PGP and thus in the pathophysiology of lung emphysema.