Bone marrow transplant-induced alterations in Notch signaling promote pathologic Th17 responses to γ-herpesvirus infection.

Idiopathic pneumonia syndrome (IPS) is a common, often fatal, complication following hematopoietic stem cell transplantation (HSCT) characterized by severe pneumonitis and interstitial fibrosis. Fully reconstituted syngeneic bone marrow transplant (BMT) mice infected with murine γ-herpesvirus-68 develop interleukin-17 (IL-17)-driven pneumonitis and fibrosis, which mimics clinical manifestations of IPS. We found CD103+ and CD11b+ dendritic cells (DCs) are selectively deficient for the Notch ligand, DLL4, following BMT and CD4+ T cells isolated from lungs and spleens of infected BMT mice display Notch signaling defects. Mice transplanted with CD4-Cre-driven dominant-negative Notch transcriptional regulator Mastermind-Like (CD4-Cre-DNMAML (CCD) mice) bone marrow displayed elevated IL-17 and transforming growth factor-ß (TGF ß) in the lungs, a further expansion of T-helper type 17 (Th17) cells, and developed more fibrosis than wild-type (WT)-BMT mice. Culture of BMT lung leukocytes with recombinant Notch ligand, DLL4, restored Notch signaling and decreased production of IL-17. Adoptive transfer of CD11c+ DCs could restore Th1 and limit Th17 in WT-BMT but not CCD-BMT mice, indicating that a specific DC/CD4+ T-cell Notch interaction modulates IL-17 production following reconstitution in syngeneic BMT mice. Given recent clinical observations showing that patients with pulmonary complications post-transplant harbor occult herpesvirus infections, these data provide mechanistic insight and suggest potential therapies for these devastating conditions.

Elevated prostaglandin E2 post-bone marrow transplant mediates interleukin-1ß-related lung injury.

Hematopoietic stem cell transplant (HSCT) treats or cures a variety of hematological and inherited disorders. Unfortunately, patients who undergo HSCT are susceptible to infections by a wide array of opportunistic pathogens. Pseudomonas aeruginosa bacteria can have life-threatening effects in HSCT patients by causing lung pathology that has been linked to high levels of the potent pro-inflammatory cytokine, interleukin-1ß (IL-1ß). Using a murine bone marrow transplant (BMT) model, we show that overexpression of prostaglandin E2 (PGE2) post-BMT signals via EP2 or EP4 to induce cyclic adenosine monophosphate (cAMP), which activates protein kinase A or the exchange protein activated by cAMP (Epac) to induce cAMP response element binding-dependent transcription of IL-1ß leading to exacerbated lung injury in BMT mice. Induction of IL-1ß by PGE2 is time and dose dependent. Interestingly, IL-1ß processing post-P. aeruginosa infection occurs via the enzymatic activity of either caspase-1 or caspase-8. Furthermore, PGE2 can limit autophagy-mediated killing of P. aeruginosa in alveolar macrophages, yet autophagy does not have a role in PGE2-mediated upregulation of IL-1ß. Reducing PGE2 levels with indomethacin improved bacterial clearance and reduced IL-1ß-mediated acute lung injury in P. aeruginosa-infected BMT mice.

Periostin regulates fibrocyte function to promote myofibroblast differentiation and lung fibrosis.

Fibrocytes are circulating mesenchymal precursors (CD45+, col 1+) recruited to fibrotic areas. Fibrocytes secrete profibrotic mediators including periostin; a matricellular protein that regulates cellular interactions with extracellular matrix (ECM) components. In bleomycin-induced fibrosis, periostin deficiency in structural or hematopoietic cells limits development of pulmonary fibrosis. To determine if hematopoietic-derived fibrocytes might secrete soluble factors to activate structural myofibroblast differentiation, wild-type (WT) fibroblasts were treated with conditioned medium from fibrocytes isolated from bleomycin-treated WT or periostin-/- mice. After 24 h we saw less α-smooth muscle actin expression in cells treated with conditioned medium from periostin-/- fibrocytes. Adoptive transfer of WT fibrocytes augmented lung fibrosis to a greater extent than transfer of fibrocytes from periostin-/- mice. In vitro analysis of fibrocytes and fibroblasts isolated from WT and periostin-/- mice treated with TGFß1 or periostin demonstrated co-regulation of mesenchymal activation and beta 1 integrin as a potential receptor for periostin on fibrocytes. Additionally, connective tissue growth factor (CTGF) mRNA expression was increased in fibrocytes treated with periostin whereas CTGF and lysl oxidase (LOX) mRNA expression was low in bleomycin-treated periostin-/- fibrocytes. These data suggest fibrocytes may augment bleomycin-induced fibrosis via secretion of periostin and other soluble factors that promote myofibroblast differentiation.

Bone marrow transplantation alters lung antigen-presenting cells to promote TH17 response and the development of pneumonitis and fibrosis following gammaherpesvirus infection.

Hematopoietic stem cell transplantation (HSCT) efficacy is limited by numerous pulmonary complications. We developed a model of syngeneic bone marrow transplantion (BMT) followed by infection with murine gamma herpesvirus-68 that results in pneumonitis and fibrosis and mimics human "noninfectious" HSCT complications. BMT mice experience increased early lytic replication, but establish viral latency by 21 days post infection. CD4 T cells in BMT mice are skewed toward interleukin (IL)-17A rather than interferon (IFN)-γ production. Transplantation of bone marrow from Il-17a(-/-) donors or treatment with anti-IL-17A neutralization antibodies at late stages attenuates pneumonitis and fibrosis in infected BMT mice, suggesting that hematopoietic-derived IL-17A is essential for development of pathology. IL-17A directly influences activation and extracellular matrix production by lung mesenchymal cells. Lung CD11c+ cells of BMT mice secrete more transforming growth factor beta-ß1, and pro-TH17 mRNAs for IL-23 and IL-6, and less TH1-promoting cytokine mRNA for IFN-γ but slightly more IL-12 mRNA in response to viral infection. Adoptive transfer of non-BMT lung CD11c-enriched cells restores robust TH1 response and suppresses aberrant TH17 response in BMT mice to improve lung pathology. Our data suggest that "noninfectious" HSCT lung complications may reflect preceding viral infections and demonstrate that IL-17A neutralization may offer therapeutic advantage even after disease onset.

Protection from pulmonary fibrosis in the absence of CCR2 signaling.

Pulmonary fibrosis can be modeled in animals by intratracheal instillation of FITC, which results in acute lung injury, inflammation, and extracellular matrix deposition. We have previously shown that despite chronic inflammation, this model of pulmonary fibrosis is lymphocyte independent. The CC chemokine monocyte-chemoattractant protein-1 is induced following FITC deposition. Therefore, we have investigated the contribution of the main monocyte-chemoattractant protein-1 chemokine receptor, CCR2, to the fibrotic disease process. We demonstrate that CCR2(-/-) mice are protected from fibrosis in both the FITC and bleomycin pulmonary fibrosis models. The protection is specific for the absence of CCR2, as CCR5(-/-) mice are not protected. The protection is not explained by differences in acute lung injury, or the magnitude or composition of inflammatory cells. FITC-treated CCR2(-/-) mice display differential patterns of cellular activation as evidenced by the altered production of cytokines and growth factors following FITC inoculation compared with wild-type controls. CCR2(-/-) mice have increased levels of GM-CSF and reduced levels of TNF-alpha compared with FITC-treated CCR2(+/+) mice. Thus, CCR2 signaling promotes a profibrotic cytokine cascade following FITC administration.

GM-CSF regulates bleomycin-induced pulmonary fibrosis via a prostaglandin-dependent mechanism.

To characterize the role of GM-CSF in pulmonary fibrosis, we have studied bleomycin-induced fibrosis in wild-type mice vs mice with a targeted deletion of the GM-CSF gene (GM-CSF-/- mice). Without GM-CSF, pulmonary fibrosis was worse both histologically and quantitatively. These changes were not related to enhanced recruitment of inflammatory cells because wild-type and GM-CSF-/- mice recruited equivalent numbers of cells to the lung following bleomycin. Interestingly, recruitment of eosinophils was absent in GM-CSF-/- mice. We investigated whether the enhanced fibrotic response in GM-CSF-/- animals was due to a deficiency in an endogenous down-regulator of fibrogenesis. Analysis of whole lung homogenates from saline- or bleomycin-treated mice revealed that GM-CSF-/- animals had reduced levels of PGE2. Additionally, alveolar macrophages were harvested from wild-type and GM-CSF-/- mice that had been exposed to bleomycin. Although bleomycin treatment impaired the ability of alveolar macrophages from wild-type mice to synthesize PGE2, alveolar macrophages from GM-CSF-/- mice exhibited a significantly greater defect in PGE2 synthesis than did wild-type cells. Exogenous addition of GM-CSF to alveolar macrophages reversed the PGE2 synthesis defect in vitro. Administration of the PG synthesis inhibitor, indomethacin, to wild-type mice during the fibrogenic phase postbleomycin worsened the severity of fibrosis, implying a causal role for PGE2 deficiency in the evolution of the fibrotic lesion. These data demonstrate that GM-CSF deficiency results in enhanced fibrogenesis in bleomycin-induced pulmonary fibrosis and indicate that one mechanism for this effect is impaired production of the potent antifibrotic eicosanoid, PGE2.

Neovascularization during venous thrombosis organization: a preliminary study.

PURPOSE: Thrombus organization after venous thromboembolism leading to recanalization occurs at a variable rate. The angiogenic chemokine interleukin-8 (IL-8) has been found in thrombus months after thrombus initiation. We hypothesize that thrombus organization involves neovascularization and leukocyte influx and that IL-8 administered at thrombus induction will promote thrombus organization. METHODS: A group of rats underwent inferior vena caval occlusive thrombosis. At thrombus induction and every 24 hours, the rats were administered IL-8 (1 microgram) or serum albumin. The rats were killed at either day 4, day 8, or day 12, and, at death, colloidal carbon was perfused via the heart. The inferior vena cava was isolated, measured, weighed, and formalin fixed. The sections were stained with anti-polymorphonuclear leukocyte antibody, the endothelial marker factor VIII-related antigen, and with hematoxylin and eosin. Thrombus neovascularization (colloidal carbon) with morphometric analysis was normalized to the total thrombus area. In addition, the rats underwent perfusion with fluorescein isothiocyanate dextran (molecular weight, 150,000) at death to correlate with colloidal carbon perfusion, and thrombus fluorescence was determined. RESULTS: Thrombus cellularity initially involved neutrophils, followed by monocytes. Significantly more neutrophils, monocytes, and cells that were defined as spindle shaped (fibroblasts and endothelial cells) were noted in the animals treated with IL-8. Neovascularization was significantly increased at day 4 in the animals treated with IL-8 versus the animals treated with serum albumin and was corroborated with a significant increase in thrombus fluorescein isothiocyanate dextran fluorescence at day 4 in the rats treated with IL-8. Colloidal carbon perfusion was noted within vascular channels without extravasation and colocalized with factor VIII-related antigen. CONCLUSION: This study shows that thrombus organization involves neovascularization and that IL-8 augments thrombus organization.

Low-dose low-molecular-weight heparin is anti-inflammatory during venous thrombosis.

PURPOSE: Venous thrombosis results in a vein wall inflammatory response initiated by thrombus. Although anticoagulation with standard heparin (SH) and low-molecular-weight heparin (LMWH) is known to limit further thrombosis, their anti-inflammatory properties are poorly defined. The anti-inflammatory properties of these heparins were studied. METHODS: Sprague-Dawley rats were divided into groups and underwent inferior vena caval (IVC) ligation just below the renal level producing IVC thrombosis. One hour before ligation, animals received subcutaneous SH or LMWH at either high or low dose; normal saline (NS) was used as control. Six hours after ligation, animals were killed, and the IVCs were analyzed for clot presence, vein wall morphometrics, and vein wall permeability (VP) to define injury. RESULTS: Animals in both low-dose groups had no measurable anticoagulation, whereas those in both high-dose groups were adequately anticoagulated. There were statistically less IVC neutrophils for all groups compared with the control group, with low-dose LMWH showing the least cells (low-dose LMWH, 16 +/- 3; high-dose LMWH, 37 +/- 10; low-dose SH, 37 +/- 6; high-dose SH, 32 +/- 9; NS control, 63 +/- 2). Similar results were noted for total inflammatory cells. The lowest VP was noted for low-dose LMWH. CONCLUSION: Although both SH and LMWH inhibited vein wall neutrophils and total inflammatory cells, low-dose LMWH was most effective limiting neutrophil extravasation and was the only intervention to decrease VP below control levels. This occurred without preventing thrombus formation or causing a state of anticoagulation. Low-dose LMWH possesses anti-inflammatory properties distinct from its anticoagulant properties.

IL-10 regulates thrombus-induced vein wall inflammation and thrombosis.

Vein wall inflammation associated with venous thrombosis is mediated by an imbalance in proinflammatory as compared with antiinflammatory molecules. We hypothesize that IL-10 is an important antiinflammatory cytokine that influences vein wall inflammation and thrombus propagation during venous thrombosis. To test this hypothesis a model of inferior vena caval thrombosis was used. Studies were performed at sacrifice 2 days after thrombus induction and included leukocyte morphometrics, myeloperoxidase activity, vein wall permeability, thrombus weight, and IL-10 ELISA analysis from the vein wall. IL-10 was elevated in the vein wall during venous thrombosis. Neutralization of IL-10 increased inflammation, while supplementation with rIL-10 demonstrated a dose- and time-dependent decrease in inflammation. Interestingly, a low 2.5-microg rIL-10 dose given at time of initiation of thrombosis most significantly decreased inflammation. Thrombus weight was importantly diminished by reconstitution of IL-10. These studies support an important role for IL-10 in the regulation of thrombus-associated inflammation and thrombosis and suggest that IL-10 could be used as a therapeutic agent in the treatment of venous thrombosis.

The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and often fatal disorder. Fibroplasia and deposition of extracellular matrix are dependent, in part, on angiogenesis. We postulated that an imbalance exists in the expression of angiogenic (IL-8) vs angiostatic (IFN-gamma-inducible protein (IP-10)) CXC chemokines, which favors net angiogenesis in IPF. To test this hypothesis, we obtained open lung biopsies either from normal patients undergoing thoracic surgery for reasons other than interstitial lung disease (control) or from patients with IPF. We found that levels of IL-8 were greater from tissue specimens of IPF patients then from those of controls. In contrast, IP-10 levels were higher from tissue specimens obtained from control subjects than from those from IPF patients. When IL-8 or IP-10 was depleted from IPF tissue specimens, tissue-derived angiogenic activity was markedly reduced or enhanced, respectively. Immunolocalization of IL-8 demonstrated that the pulmonary fibroblast (PF) of IPF lung was the predominant cellular source of IL-8. Isolated PF from IPF patients constitutively produced more IL-8 and less IP-10 than control PF. Conditioned media from IPF-PFs demonstrated constitutive angiogenic activity that was attributable, in part, to IL-8. Depletion of IP-10 from IPF-PF CM resulted in an increase in corneal neovascularization. These findings support the notion that IL-8 and IP-10 are important factors that regulate angiogenic activity in IPF.

Anti-P-selectin antibody decreases inflammation and thrombus formation in venous thrombosis.

PURPOSE: Venous thrombosis and inflammation are interrelated. P-selectin contributes to activation of leukocyte-mediated inflammation. Therefore, we hypothesized that the neutralization of P-selectin would decrease vein wall inflammation and thrombosis. METHODS: Twelve baboons underwent infrarenal inferior vena caval balloon occlusion to induce thrombosis. Two groups of four baboons received neutralizing intravenous anti-P-selectin antibody (PSab) GA6 or CY1748 before occlusion and at days 2 and 4. Four baboons received saline control injections. One baboon per group was killed at days 2, 6, and 13, and at 2 months. Analysis included phlebography, ultrasound, gadolinium (Gd)-enhanced magnetic resonance venography (reflecting vein wall inflammation), and histologic, morphometric, and protein evaluation of the vein wall. Thrombus presence or absence was assessed. RESULTS: By day 2 in PSab baboons, vein wall Gd enhancement was decreased in the mid-inferior vena cava and the right iliac vein (p < 0.05; GA6 vs control baboons), normalizing by 2 months. The mid-inferior vena cava revealed fewer neutrophils and total leukocytes in PSab baboons; however, for GA6 in the right iliac vein these decreases were not present despite the absence of Gd enhancement; they were decreased with CY1748. PSab baboons demonstrated significantly less thrombus than control baboons (p < 0.01, GA6 and CY1748 vs control baboons). CONCLUSIONS: Anti-P-selectin antibody decreases vein wall inflammation and thrombus formation. Inhibition of P-selectin may be useful in venous thrombosis prophylaxis.

Bronchoalveolar lavage neutrophilia is associated with obliterative bronchiolitis after lung transplantation: role of IL-8.

Obliterative bronchiolitis (OB) is a devastating complication in lung transplantation. We postulated that the pathogenesis of OB is mediated, in part, by neutrophils. We serially collected bronchoalveolar lavage (BAL) fluid from lung transplant recipients. Patients were divided into two groups depending on the presence or absence of OB. Samples from patients who never developed OB were further divided according to whether rejection was present. These samples were labeled healthy or rejection. Samples from patients who developed OB were divided according to whether the sample was obtained before (future OB) or at the time of diagnosis of OB (OB). The OB group, as compared with the healthy and rejection group, had significantly elevated neutrophil counts (3.9 x 10(5) +/- 1.8 x 10(5) vs 0.3 x 10(5) +/- 0.07 x 10(5) and 0.4 x 10(5) +/- 0.1 x 10(5), respectively, p < 0.01 for both) and levels of IL-8 (3131 +/- 1468 pg/ml vs 240 +/- 62 pg/ml and 172 +/- 47 pg/ml, p < 0.01 for both). Furthermore, we demonstrated immunolocalization of IL-8 associated with alpha smooth muscle actin-positive cells in the peribronchial region of OB. To confirm that the IL-8 present in BAL fluid from patients with OB was bioactive, we performed neutrophil chemotaxis experiments that showed that IL-8 accounted for a significant amount of the neutrophil chemotactic activity. We also found a trend toward higher levels of neutrophils and IL-8 in BALs from the future OB as compared with the healthy group (7.1 x 10(4) +/- 4.2 x 10(4) vs 3.4 x 10(4) +/- 0.7 x 10(4) and 500 +/- 306 pg/ml vs 240 +/- 62 pg/ml). In conclusion, we have provided the novel observation that in lung transplant recipients with OB, neutrophilia is present and highly correlated with the presence of IL-8.

Neutrophils are the initial cell type identified in deep venous thrombosis induced vein wall inflammation.

Venous thrombosis and inflammation are interrelated. The authors hypothesized that inferior vena cava thrombosis results in a predictable vein wall inflammatory response, characterized by early neutrophil infiltration. Thrombosis was induced in rats by placement of an inferior vena cava ligature with branch ligation. Animals were killed at baseline, 6 hrs, day 2, and day 6. Analysis included vein wall morphometrics, myeloperoxidase activity, and fluorescence activated cell sorting. At 6 hrs, there was an increase in neutrophils and lymphocytes as compared to sham animals (p < 0.0001 for neutrophils, p < 0.05 for lymphocytes). By day 2, only neutrophils were elevated in the experimental groups (experimental = 75.5 cells/5 high power fields vs. 9.6 cells/ 5 high power fields in shams, p < 0.0001). Myeloperoxidase activity in the experimental group was greater than shams on day 2(34.7 delta optical density/min vs. 5.9 delta optical density/ min, p < 0.0001). Fluorescence activated cell sorting of the neutrophil marker at 6 hrs confirmed the increase in neutrophils (experimental = 63.1%, shams = 39.1%, p < 0.0001), and peaked on day 2 (71.9%). This study suggests that 1) neutrophils are elevated early during the inflammatory response due to thrombus initiation, and 2) neutrophils, because of their early predominance, likely contributed to vein wall injury during venous thrombosis.

P-selectin and TNF inhibition reduce venous thrombosis inflammation.

Venous thrombosis induces a detrimental inflammatory response in the vein wall. The cytokine tumor necrosis factor-alpha (TNF) and the adhesion molecules, selectins, have been found to be important in mediating inflammatory cell stimulation and leukocyte-endothelial cell adhesion, respectively. This study assesses the role of TNF and P-selectin in the inflammatory events associated with venous thrombosis. Rats were passively immunized with neutralizing anti-TNF serum alone, anti-TNF plus anti-P-selectin antibody, anti-P-selectin antibody alone, control serum, or control anti-P-selectin antibody. Antibodies or control sera were given prior to occlusion and at Days 2 and 4 postocclusion. Rats were sacrificed at Days 1-6 and Day 13 after occlusion for inferior vena caval (IVC) wall histopathology and TNF analysis. Differences in the extent of inflammatory cell infiltrate into the vein wall were found on Days 2, 6, and 13. TNF levels were elevated in the vein wall of the three groups not given anti-TNF antibody. The levels of TNF at Day 6 positively correlated with both total inflammatory cell (r = 0.53, P < 0.05) and neutrophil presence (r = 0.72, P < 0.01). The lowest IVC wall neutrophil and total inflammatory cell count at Days 2 and 6 and the lowest neutrophil count at Day 13 were found in the anti-TNF plus anti-P-selectin antibody group. Monocyte influx was also inhibited at Day 13 in this group. These results suggest a role for combined neutralization of TNF and P-selectin in the attenuation of inflammation induced by venous thrombosis.

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.

Increased interleukin-1 receptor antagonist in idiopathic pulmonary fibrosis. A compartmental analysis.

Idiopathic pulmonary fibrosis (IPF) is a poorly understood interstitial disease that usually proves refractory to therapy and results in irreversible tissue scarring and pulmonary dysfunction. Previous investigations have suggested a number of possible mediators of inflammation and fibrosis that typify IPF. We report increases in lung interleukin-1 receptor antagonist protein (IRAP) content in patients with IPF, as compared with normal control subjects. Importantly, this increase in IRAP was not accompanied by concomitant increases in interleukin-1 beta (IL-1 beta), resulting in a local environment that may be profibrotic. Tissue homogenates and bronchoalveolar lavage fluid from patients with IPF both demonstrate elevated IRAP content compared with that in normal subjects. Immunohistochemical staining and in situ hybridization localize IRAP to hyperplastic type II pneumocytes, macrophages, and local stromal cells. Finally, in vitro studies utilizing fibroblasts isolated from patients with IPF demonstrated no difference in constitutive IRAP production compared with that in normal subjects, but they revealed an exaggerated response to stimulation with transforming growth factor-beta (TGF-beta). These findings suggest that the fibrotic tissue changes of IPF and possibly other chronic interstitial lung diseases may result in part from the local effects of IRAP, and they also demonstrate that pulmonary nonimmune cells may influence local tissue changes through the elaboration of IRAP.

Venous thrombosis-associated inflammation and attenuation with neutralizing antibodies to cytokines and adhesion molecules.

Thrombosis and inflammation are closely related. However, the response of the vein wall to venous thrombosis has been poorly documented. This study examines the hypothesis that venous thrombosis is associated with an inflammatory response in the vein wall. In a rat model of inferior vena caval thrombosis, vein wall was temporally examined for inflammation by assessment of histopathology, leukocyte morphometrics, and cytokine levels. Animals were killed 1 hour and 1, 3, and 6 days after thrombus induction. Our findings demonstrated an early (day 1) neutrophil infiltration into the vein wall followed by a later (days 3 and 6) monocyte/macrophage and lymphocyte response. Cytokines were elevated only under conditions of venous thrombosis. Levels of epithelial neutrophil activating protein-78 (ENA-78), tumor necrosis factor-alpha (TNF), interleukin-6, and JE/monocyte chemoattractant protein-1 (JE/MCP-1) increased over the 6-day period, while macrophage inflammatory protein-1 alpha (MIP-1 alpha) peaked at day 3 after thrombus induction. Additionally, rats were passively immunized with neutralizing antibodies to TNF, ENA-78, MIP-1 alpha, JE/MCP-1, intercellular adhesion molecule-1 (ICAM-1), and CD18 compared with control antibodies. The most effective antibody early after thrombus induction for attenuating vein wall neutrophil extravasation was anti-TNF (P < .01). The monocyte/macrophage extravasation was inhibited most by anti-ICAM-1 followed by anti-TNF (P < .01). These findings demonstrate that venous thrombosis is associated with significant vein wall inflammation that is partially inhibited by neutralizing antibodies to cytokines and adhesion molecules.

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.

Production and function of murine macrophage inflammatory protein-1 alpha in bleomycin-induced lung injury.

We investigated the role of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in bleomycin-induced lung injury, a model of interstitial lung disease. Bleomycin stimulates a T cell-dependent pulmonary inflammatory response characterized by an increase in leukocyte infiltration, fibroblast proliferation, and collagen synthesis. Intratracheal challenge of CBA/J mice with bleomycin resulted in a significant time-dependent increase in MIP-1 alpha protein levels both in whole-lung homogenates and bronchoalveolar lavage fluid. The kinetics of MIP-1 alpha expression were biphasic, with the first peak occurring at 2 days postinstillation and the second peak at 16 days. These levels of Ag expression temporally correlated with the accumulation of granulocytes, lymphocytes, and mononuclear phagocytes in the lung. In addition, immunohistochemical staining identified alveolar macrophages and bronchial epithelial cells as the primary cellular sources of MIP-1 alpha production. Interestingly, passive immunization of bleomycin-challenged mice with anti-MIP-1 alpha Abs significantly reduced pulmonary mononuclear phagocyte accumulation and fibrosis. These experiments establish that MIP-1 alpha protein is expressed in the lungs of bleomycin-treated mice and provide evidence that MIP-1 alpha promotes leukocyte accumulation and activation. Furthermore, these findings support the notion that leukocyte accumulation and activation are linked to fibrosis.