Inhibit Leukocyte Accumulation Research Articles

Abstract 031: A Ligand-specific Blockade of the Integrin Mac-1 Selectively Targets Pathologic Vascular Inflammation While Maintaining Protective Host-defense

Background: Integrins, such as Mac-1 (alphaMbeta2, CD11b/CD18), drive myeloid cell recruitment and inflammation in cardiovascular disease. Although integrins have generated interest as therapeutic targets in acute and chronic inflammation, they also contribute to host defense, regeneration, and haemostasis. To overcome these limitations, we have designed a novel antibody that targets a distinct region within the Mac-1 major ligand-binding (I-) domain required for binding of its ligand CD40L. Here, we describe the consequences of a ligand-specific inhibition and its efficacy in mice. Methods and Results: To generate an antibody specifically targeting the interaction of Mac-1 with CD40L, mice were immunized with a peptide containing the CD40L-binding motif EQLKKSKTL. Antibody clones were screened and tested for specificity. One clone, termed anti-M7, showed specific peptide binding and blocked the binding of the activated, open conformation of Mac-1 to CD40L, but not to alternative Mac-1 ligands suggesting ligand specificity. Anti-M7 highly effectively limited leukocyte recruitment in vitro and to the mesentery and the omentum in intravital confocal microscopy. While conventional anti-Mac-1 antibodies induced outside-in activation of the integrin, anti-M7 did not provoke MAP-kinase dependent activation and expression of inflammatory cytokines in macrophages. Following cecal-ligation and puncture (CLP) sepsis in mice, a conventional anti-Mac-1 antibody inhibited leukocyte accumulation, but potentiated bacteremia and limited overall survival. In contrast, anti-M7 limited peritoneal leukocyte accumulation, reduced bacterial titers, and improved survival during CLP. Conclusion: We present proof-of-concept demonstrating selective inhibition of the interaction between the leukocyte integrin Mac-1 and CD40L by a novel monoclonal antibody. This antibody shows several advantages over conventional anti-integrin therapy, and prevented inflammation and sepsis-related mortality in mice. This novel approach gives new insight into ligand-specific integrin pathways and merits further evaluation as a selective anti-inflammatory therapy in cardiovascular disease.

Recombinant Human Soluble Thrombomodulin Attenuates Hepatic Ischemia and/or Reperfusion Injury by Inhibiting Leukocyte Accumulation in Mice With Normal and Fatty Liver

In an attempt to increase the number of donor livers, there has been an increased use of marginal donor livers, such as steatotic (fatty) livers that increase susceptibility to ischemia and reperfusion injury (IRI). Inflammatory cell accumulation has a greater role in IRI in steatotic liver than in normal liver. Although the recombinant human soluble thrombomodulin (rhsTM) attracts attention as a new treatment for disseminated intravascular coagulation, the therapeutic efficacy of rhsTM in hepatic IRI remains uncertain, especially in fatty livers. We aimed to demonstrate the effect of rhsTM on hepatic IRI using well-established in vivo experimental models with steatotic liver. MethodsC57/BL6 mice were divided into 2 groups: normal liver (NL) group and fatty liver (FL) group, in which the steatotic liver was induced by high-fat diet for 9 weeks. The mice in the NL and FL groups were premedicated with venous injection of rhsTM (TM) or saline (Control) as control groups. All 4 groups (NL-Control vs NL-TM, FL-Control vs FL-TM) were subjected to partial hepatic warm ischemia followed by reperfusion. ResultsrhsTM significantly attenuated liver injury in the FL group as well as the NL group, as evidenced by transaminase levels and histologic finding after hepatic IRI. rhsTM remarkably decreased the accumulation of inflammatory cells, such as macrophages and neutrophils, in both NL and FL tissue after IRI. Furthermore, rhsTM depressed mRNA and protein expressions of adhesion molecules such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 in both NL and FL groups after IRI. ConclusionOur results demonstrate that rhsTM has a protective effect on fatty liver as well as normal liver after hepatic IRI. They also suggest that rhsTM contributes to attenuation of leukocyte accumulation caused by depressing expressions of adhesion molecules that facilitate accumulation of leukocytes in liver tissue in hepatic IRI.

Evaluation of innate immune responses in bovine forestomachs

Previous studies had indicated an active role of bovine forestomachs in the response to alimentary disorders as well as to inflammatory and infectious processes in both the gastro-intestinal (GI) tract and elsewhere. We investigated the potential of bovine forestomachs to receive, elaborate and produce signals and mediators of the innate immune response. Indeed, we detected the expression of Toll IL-1R8/single Ig IL-1-related receptor (TIR8/SIGIRR) and other receptors and cytokines, such as Toll-like receptor (TLR)4, interleukin (IL)-1β, IL-10 and Caspase-1 in the forestomach walls of healthy cows. Their presence suggests an active role of forestomachs in inflammatory disorders of the GI tract and other body compartments. Moreover, interferon (IFN)-γ was revealed in ruminal content. We confirmed and further characterized the presence of leukocytes in the rumen fluids. In particular, T-, B-lymphocytes and myeloid lineage cells were detected in the ruminal content of both rumen-fistulated heifers and diseased cows. An acidogenic diet based on daily supplements of maize was shown to inhibit leukocyte accumulation, as opposed to a control, hay-based diet, with or without a soy flour (protein) supplement. On the whole, results indicate that bovine forestomachs can receive and elaborate signals for the immune cells infiltrating the rumen content or other organs. Forestomachs can thus participate in a cross-talk with the lymphoid tissues in the oral cavity and promote regulatory actions at both regional and systemic levels; these might include the control of dry matter intake as a function of fundamental metabolic requirements of ruminants.

Intratracheal Antitumor Necrosis Factor‐α Antibody Attenuates Lung Tissue Damage Following Cardiopulmonary Bypass

The aim of this study is to investigate the protective effect and underlying mechanism of antitumor necrosis factor-α antibody (TNF-α Ab) on lung tissue injury after cardiopulmonary bypass (CPB). Twenty-eight healthy New Zealand white rabbits were randomly divided into four groups. Group I received only an open chest operation. Groups II-IV all received CPB. Furthermore, groups III and IV received post-CPB endotracheal intubation with phosphate buffered saline or TNF-α Ab (2400 pg/kg), respectively. Perioperative blood neutrophil count, TNF-α level, and malondialdehyde (MDA) levels were determined in both the right and left atriums. Lung water content, TNF-α messenger RNA, protein, apoptosis in situ, and pathomorphological changes were also measured. The results show that TNF-α Ab can significantly inhibit leukocyte accumulation, reduce secretion of TNF-α and MDA, decrease lung tissue apoptosis, and attenuate post-CPB pathomorphological changes. TNF-α Ab administration, however, cannot suppress the expression of TNF-α, suggesting that the protective effects of TNF-α Ab originate from inhibiting the numerous biological functions of TNF-α. Intratracheal TNF-α Ab administration demonstrates a notable protective effect against lung injury after CPB.

Purification of a Chitin-Binding Protein from Moringa oleifera Seeds with Potential to Relieve Pain and Inflammation

Moringa oleifera Lam. is a perennial multipurpose tree that has been successfully used in folk medicine to cure several inflammatory processes. The aim of this study was to purify and characterize a chitin-binding protein from Moringa oleifera seeds, named Mo-CBP4, and evaluate its antinociceptive and anti-inflammatory effects in vivo. The protein was purified by affinity chromatography on chitin followed by ion exchange chromatography. Acetic acid-induced abdominal constrictions assay was used for the antinociceptive and anti-inflammatory activity assessments. Mo-CBP4 is a glycoprotein (2.9% neutral carbohydrate) composed of two protein subunits with apparent molecular masses of 28 and 18 kDa (9 kDa in the presence of reducing agent). The intraperitoneal injection of Mo-CBP4 (3.5 and 10 mg/kg) into mice 30 min before acetic acid administration potently and significantly reduced the occurrence of abdominal writhing in a dose dependent manner by 44.7% and 100%, respectively. In addition, the oral administration of the protein (10 mg/kg) resulted in 18% and 52.8% reductions in abdominal writhing when given 30 and 60 min prior to acetic acid administration, respectively. Mo-CBP4, when administered by intraperitoneal route, also caused a significant and dose-dependent inhibition of peritoneal capillary permeability induced by acid acetic and significantly inhibited leukocyte accumulation in the peritoneal cavity. In conclusion, this pioneering study describes that the chitin-binding protein Mo-CBP4, from M. oleifera seeds, exhibits anti-inflammatory and antinociceptive properties and scientifically supports the use of this multipurpose tree in folk medicine.

A role for MC3R in modulating lung inflammation

In this study we set out to ascertain whether melanocortin peptides could be potential therapeutic agents in allergic and non-allergic models of lung inflammation by identifying the receptor(s) involved using a molecular, genetic and pharmacological approach. Western blot analyses revealed expression of the melanocortin receptor (MCR) type 1 and 3 on alveolar macrophages from wild-type mice. Alveolar macrophage incubation, with the selective MC3R agonist [D-TRP 8]- γ-MSH and pan-agonist α-MSH but not the selective MC1R agonist MS05, led to an increase in cAMP in wild-type macrophages. This increase occurred also in macrophages taken from recessive yellow (e/e; bearing a mutant and inactive MC1R) mice but not from MC3R-null mice. In an allergic model of inflammation, the pan-agonist α-MSH and selective MC3R agonist [D-TRP 8]- γ-MSH displayed significant attenuation of both eosinophil and lymphocyte accumulation but not IL-5 levels in wild-type and recessive yellow e/e mice. However in MC3R-null mice, α-MSH failed to cause a significant inhibition in these parameters, highlighting a preferential role for MC3R in mediating the anti-inflammatory effects of melanocortins in this model. Utilising a non-allergic model of LPS-induced lung neutrophilia, the pan-agonist α-MSH and selective MC3R agonist [D-TRP 8]- γ-MSH displayed significant attenuation of neutrophil accumulation and inhibition of TNF- α release. Thus, this study highlights that melanocortin peptides inhibit leukocyte accumulation in a model of allergic and non-allergic inflammation and this protective effect is associated with activation of the MC3R. The inhibition of leukocyte accumulation is via inhibition of TNF- α in the non-allergic model of inflammation but not IL-5 in the allergic model. These data have highlighted the potential for selective MC3R agonists as novel anti-inflammatory therapeutics in lung inflammation.

Neuroprotection by Recombinant Thrombomodulin

We examined whether recombinant human soluble thrombomodulin (rhs-TM) reduces compression trauma-induced spinal cord injury through protein C activation in rats. Administration of rhs-TM, either before or after the induction of spinal cord injury (SCI), markedly reduced the resulting motor disturbances. However, neither rhs-TM pretreated with an anti-rhs-TM monoclonal antibody (MAb) F2H5, which inhibits thrombin binding to rhs-TM, nor those pretreated with MAb R5G12, which selectively inhibits protein C activation by rhs-TM, prevented the motor disturbances. Intramedullary hemorrhages, observed 24 h after trauma, were significantly reduced in animals given rhs-TM. The increase in the tissue levels of tumor necrosis factor-alpha (TNF-alpha), TNF-alpha mRNA expression, and the accumulation of leukocytes in the damaged segment of the spinal cord were significantly inhibited in animals receiving rhs-TM, but these effects were not observed following administration of rhs-TM pretreated with MAb R5G12 or MAb F2H5. Leukocytopenia and activated protein C all produced effects similar to those of rhs-TM. These findings suggest that rhs-TM prevents compression trauma-induced SCI by inhibiting leukocyte accumulation by reducing the expression of TNF-alpha mRNA and such therapeutic effects of rhs-TM could be dependent on its protein C activation capacity. Findings further suggest that thrombomodulin can be implicated not only in the coagulation system but in regulation of the inflammatory response.

Salicylates, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 (p50) of nuclear factor Kb (NFκb)

Salicylates, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 (p50) of nuclear factor Kb (NFκb)

Leukocyte recruitment in the cerebrospinal fluid of mice with experimental meningitis is inhibited by an antibody to junctional adhesion molecule (JAM).

The mechanisms that govern leukocyte transmigration through the endothelium are not yet fully defined. Junctional adhesion molecule (JAM) is a newly cloned member of the immunoglobulin superfamily which is selectively concentrated at tight junctions of endothelial and epithelial cells. A blocking monoclonal antibody (BV11 mAb) directed to JAM was able to inhibit monocyte transmigration through endothelial cells in in vitro and in vivo chemotaxis assays. In this study, we report that BV11 administration was able to attenuate cytokine-induced meningitis in mice. The intravenous injection of BV11 mAb significantly inhibited leukocyte accumulation in the cerebrospinal fluid and infiltration in the brain parenchyma. Blood-brain barrier permeability was also reduced by the mAb. We conclude that JAM may be a new target in limiting the inflammatory response that accompanies meningitis.

Gastric prostacyclin (PGI 2) prevents stress-induced gastric mucosal injury in rats primarily by inhibiting leukocyte activation

We investigated whether, in rats, gastric prostacyclin (PGI 2) prevented gastric mucosal injury that was induced by water-immersion restraint stress by inhibiting leukocyte activation. Gastric levels of 6-keto-PGF 1α, a stable metabolite of PGI 2, increased transiently 30 min after stress, followed by a decrease to below the baseline 6–8 h after stress. Gastric mucosal blood flow decreased to ∼40% of the baseline level 8 h after stress. Myeloperoxidase activity was significantly increased 8 h after stress. Treatment with indomethacin before stress inhibited the increase in 6-keto-PGF 1α levels and markedly reduced mucosal blood flow. It also markedly increased leukocyte accumulation and mucosal lesion formation. Iloprost, a stable PGI 2 analog, inhibited the indomethacin-induced decrease in mucosal blood flow, mucosal lesion exacerbation, and increase in leukocyte accumulation. Nitrogen mustard-induced leukocytopenia inhibited the indomethacin-associated lesion exacerbation and the increase in leukocyte accumulation, but not the decreases in mucosal blood flow. These observations indicate that gastric PGI 2 decreases gastric mucosal lesion formation primarily by inhibiting leukocyte accumulation.

Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of NFkappaB.

The antiinflammatory action of aspirin generally has been attributed to direct inhibition of cyclooxygenases (COX-1 and COX-2), but additional mechanisms are likely at work. These include aspirin's inhibition of NFkappaB translocation to the nucleus as well as the capacity of salicylates to uncouple oxidative phosphorylation (i.e., deplete ATP). At clinically relevant doses, salicylates cause cells to release micromolar concentrations of adenosine, which serves as an endogenous ligand for at least four different types of well-characterized receptors. Previously, we have shown that adenosine mediates the antiinflammatory effects of other potent and widely used antiinflammatory agents, methotrexate and sulfasalazine, both in vitro and in vivo. To determine in vivo whether clinically relevant levels of salicylate act via adenosine, via NFkappaB, or via the "inflammatory" cyclooxygenase COX-2, we studied acute inflammation in the generic murine air-pouch model by using wild-type mice and mice rendered deficient in either COX-2 or p105, the precursor of p50, one of the components of the multimeric transcription factor NFkappaB. Here, we show that the antiinflammatory effects of aspirin and sodium salicylate, but not glucocorticoids, are largely mediated by the antiinflammatory autacoid adenosine independently of inhibition of prostaglandin synthesis by COX-1 or COX-2 or of the presence of p105. Indeed, both inflammation and the antiinflammatory effects of aspirin and sodium salicylate were independent of the levels of prostaglandins at the inflammatory site. These experiments also provide in vivo confirmation that the antiinflammatory effects of glucocorticoids depend, in part, on the p105 component of NFkappaB.

Induction of Acute Inflammation In Vivo by Staphylococcal Superantigens. II. Critical Role for Chemokines, ICAM-1, and TNF-α

AbstractSuperantigens such as staphylococcal enterotoxin A and B (SEA and SEB) activate the immune system by stimulating a large proportion of T lymphocytes through specific Vβ regions of the TCR and activating macrophages by binding to MHC class II molecules. While the mechanisms by which superantigens activate T lymphocytes have been elucidated, their role in the generation of local immune responses to bacterial invasion is still unclear. In this study we have examined the ability of the superantigens SEA and SEB to elicit an inflammatory reaction in vivo, in s.c. air pouches in the mouse. Upon injection into the s.c. air pouch, the two superantigens stimulated a time-dependent increase in the number of leukocytes appearing in the pouch exudate. The leukocytes migrating into the pouch exudate were predominantly neutrophils, with some mononuclear phagocytes and eosinophils present. No T lymphocytes were detected either in the pouch lining tissue or in the exudate cells. Injection of SEA resulted in increased ICAM-1 expression, as detected by immunohistochemistry, on endothelial cells in the tissue surrounding the air pouch and accumulation of TNF-α and the chemokines macrophage inflammatory protein-2 (MIP-2), MIP-1α, and JE in the pouch exudate. In addition, pretreatment of mice with Abs raised against ICAM-1, TNF-α, MIP-2, MIP-1α, KC, or JE inhibited leukocyte accumulation induced by SEA. These data demonstrate that bacterial superantigens may promote inflammation at extravascular sites in vivo, and that this response is secondary to the generation of inflammatory mediators, including chemokines.

Antithrombin III (AT III) prevents LPS-induced pulmonary vascular injury: novel biological activity of AT III.

Acute respiratory distress syndrome (ARDS) adversely affects the outcome of patients with disseminated intravascular coagulation (DIC) associated with sepsis. To determine whether antithrombin III (AT III) is useful for the treatment of ARDS in sepsis, we evaluated the effect of AT III on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. Although the intravenous administration of AT III (250 U/kg) prevented LPS-induced pulmonary accumulation of leukocytes, increases in pulmonary vascular permeability, and coagulation abnormalities, inactivated factor Xa, a selective inhibitor of thrombin generation, did not prevent such events other than the coagulation abnormalities. AT III promotes the endothelial release of prostacyclin by interacting with cell surface glycosaminoglycans in vivo. Trp49-modified AT III, which lacks affinity for heparin, did not prevent LPS-induced pulmonary vascular injury. Plasma levels of 6-keto-prostaglandin F1alpha were markedly increased in rats after the administration of LPS and significantly decreased in the LPS-treated rats administered Trp49-modified AT III, but not altered in those LPS-treated rats receiving AT III. Preventive effects of AT III were not observed in rats pretreated with indomethacin, which inhibits prostacyclin biosynthesis. Prostacyclin prevents LPS-induced pulmonary vascular injury by inhibiting leukocyte accumulation in the lungs. These observations strongly suggest that AT III prevents pulmonary vascular injury induced by LPS by promoting the endothelial release of prostacyclin, a potent inhibitor of leukocyte activation.

Glucocorticoid receptor regulates expression of L-selectin and CD11/CD18 on human neutrophils.

Recent studies have raised the hypothesis that glucocorticoids could diminish the ability of endothelial cells to direct leukocyte traffic into inflamed tissues by inhibiting expression of the adhesion molecules endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1. The aim of the present study was to investigate whether glucocorticoids also regulate the expression of L-selectin and CD11/CD18 integrins on human neutrophil granulocytes. Incubation of human whole blood with platelet-activating factor (PAF, 1 mumol/L) evoked downregulation of L-selectin and upregulation of CD11/CD18 adhesion receptors on neutrophils as measured by flow cytometry. While dexamethasone (0.1 nmol/L to 100 mumol/L) did not affect expression of adhesion molecules on resting neutrophils, it attenuated the PAF-induced changes in L-selectin and CD18 expression in a time- and concentration-dependent fashion with IC50 values of 31 and 13 nmol/L, respectively. These effects of dexamethasone were completely aborted by RU-486 (10 mumol/L), which blocks transcriptional activation of the glucocorticoid receptor, and by the protein synthesis inhibitor cycloheximide (35.5 mumol/L). Dexamethasone, up to a concentration of 1 mumol/L, neither affected significantly the release of granule enzymes nor interfered with PAF binding to its membrane receptors. Our results show that glucocorticoids at clinically relevant concentrations exert specific actions on expression of adhesion molecules on activated neutrophils, which are mediated through ligation of glucocorticoid receptors and induction of protein synthesis, and suggest a novel mechanism by which anti-inflammatory corticosteroids may inhibit leukocyte accumulation.

Laminin peptide ameliorates brain injury by inhibiting leukocyte accumulation in a rat model of transient focal cerebral ischemia.

Postischemic cerebral inflammation has been reported to contribute to ischemic brain damage. During inflammation, constituents of the extracellular matrix such as fibronectin and laminin are recognized by certain integrins or proteoglycans and play an important role in the cell adhesion process. The purpose of this study was to evaluate the efficacy of peptides derived from laminin on leukocyte accumulation, infarct size, and neurological outcome in rats subjected to 1 h of cerebral ischemia and 48 h of reperfusion. Forty-four animals were included in this study: transient ischemia without treatment (Group I), treatment with TG-1 peptide (Group II), GD-1 peptide (Group III), and GD-6 peptide (Group IV). Group II showed a significant reduction of the leukocyte accumulation (p < 0.001) and infarct size (p = 0.015) when compared with Group I. The neurological grade of Group II was also significantly better than in Group I at 48 h after reperfusion (p = 0.012). Based on these data, which are the first to explore the therapeutic potential of this peptide in cerebral ischemia, laminin peptide may offer a novel therapeutic approach to allaying injury in ischemic stroke.

Is nitric oxide involved in the regulation of the rat testicular vasculature?

Using immunohistochemistry, endothelial nitric oxide synthase (NOS), and neuronal NOS were localized in the endothelium of rat testicular arteries and in Leydig cells, respectively. NADPH-diaphorase activity, indicating NOS activity, however, was present only in endothelial cells. In order to examine the role of nitric oxide (NO) in the regulation of rat testicular vasculature, intact and hCG-pretreated (50-100 IU hCG given s.c. 6 h earlier) animals were given injections of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), 10 mg/kg i.v.). In all rats this resulted in a major increase in blood pressure. In intact, unstimulated animals, testicular vascular resistance was unaffected, and testicular blood flow consequently increased. In hCG-treated animals, in contrast, vascular resistance increased in an hCG dose-related way. L-NAME treatment also increased the hCG-induced accumulation of polymorphonuclear leukocytes in testicular venules. Treatment with N(G)-nitro-D-arginine methyl ester (D-NAME, 10 mg/kg i.v.), an inactive isomer of L-NAME, had no effect on the testicular vasculature. The study suggests that NO plays only a limited role in the regulation of testicular blood flow under basal conditions. After hCG treatment, however, NOS activity appears to be increased (increased endothelial NADPH-diaphorase staining), suggesting that NO in this situation is of importance to increase blood flow and to inhibit leukocyte accumulation.

Reduction of brain injury using heparin to inhibit leukocyte accumulation in a rat model of transient focal cerebral ischemia. II. Dose-response effect and the therapeutic window.

The administration of massive doses of heparin has been demonstrated to reduce reperfusion injury. The authors have found that heparin's antileukocyte adhesion property may play a more important role than its anticoagulant property in preventing ischemia and reperfusion injury. Although the administration of massive doses of heparin has been demonstrated to reduce brain injury after ischemia and reperfusion, the optimum dosage and timing for heparin administration remain unknown. The purpose of this study was to evaluate the dose-response effect and determine the time during which heparin must be administered to inhibit leukocyte accumulation, reduce infarct size, and improve neurological outcome in rats subjected to 1 hour of cerebral ischemia and 48 hours of reperfusion. Forty-nine animals were included in the study. The animals receiving commercial unfractionated heparin at a total dose of 2.67 to 4 mg/kg showed a significant inhibition of leukocyte accumulation, reduced infarct size, and lessened neurological dysfunction 48 hours after reperfusion (p < 0.05) when compared to untreated animals. The animals receiving unfractionated heparin within 3 hours after reperfusion also showed significantly better results than untreated animals. These data indicate that standard doses of heparin prevent reperfusion injury, and relatively late postischemic administration of heparin also is effective in brain protection. These findings may have therapeutic potential as an adjunct to thrombolytic therapy and possibly for other perfusion deficiencies with leukocyte-endothelial interaction. In view of these encouraging experimental findings, the clinical application of heparin administration after ischemia and reperfusion warrants serious consideration.

Reduction of brain injury using heparin to inhibit leukocyte accumulation in a rat model of transient focal cerebral ischemia. I. Protective mechanism.

Heparin has long been established as an anticoagulant. Although heparin has been demonstrated to reduce brain injury after ischemia and reperfusion, its mechanism of action remains unknown. Recent investigations reveal that it can modulate biological processes such as binding to adhesion receptors on endothelial cells and leukocytes. The authors hypothesized that heparin's protective effect is closely related to its antileukocyte adherence property. They evaluated the efficacy of sulfated polysaccharides (unfractionated heparin, low-molecular-weight heparin, heparan sulfate, chondroitin sulfate C, and dextran sulfate) on leukocyte accumulation, infarction size, and neurological outcome after transient focal cerebral ischemia in rats subjected to 1 hour of ischemia and 48 hours of reperfusion. Forty-nine animals were included in the study. The animals receiving unfractionated heparin or dextran sulfate showed a significant reduction in leukocyte accumulation, infarct size, and neurological dysfunction 48 hours after reperfusion (p < 0.05) when compared to untreated animals. The animals receiving unfractionated heparin also showed significantly better results than the animals receiving an equivalent anticoagulant dose of low-molecular-weight heparin. These data indicate that heparin's antileukocyte property plays a more important role than its anticoagulant ability in neuronal protection. The relative potency of the sulfated polysaccharides tested in leukocyte depletion was closely related to their degree of sulfation. Thus, in addition to demonstrating the potential efficacy of heparin as a therapeutic agent for ischemia and reperfusion injury by the prevention of leukocyte accumulation, the results also serve as a basis for studying important cellular and molecular events that contribute to tissue damage.

Endogenous glucocorticoids inhibit neutrophil recruitment to inflammatory sites in cholestatic rats.

Since glucocorticoids have been shown to inhibit leukocyte accumulation to inflammatory sites (6,9) and endogenous glucocorticoid levels are elevated in our cholestatic rat model, we investigated their role during acute inflammation. Sprague-Dawley rats (150-200 g) were either bile duct resected (BDR) or sham resected (sham). Five days later, a 2% carrageenan solution in saline was injected subcutaneously into preformed air pouches on their backs. Animals were killed 5 h later, and inflammatory response was quantitated by measuring the exudate volume, cell count, and myeloperoxidase (mainly in neutrophils) activity. We also pretreated BDR/sham rats with RU-486 (2 mg/kg ip), a glucocorticoid receptor antagonist, 1 h before carrageenan injection. BDR rats exhibited an impaired inflammatory response reflected by 20, 52, and 42% decreases in exudate volume, cell count, and myeloperoxidase activity, respectively (all P < 0.05). RU-486 treatment significantly increased the inflammatory response in BDR rats (to sham levels), but not in sham rats. These results suggest that endogenous glucocorticoid suppresses the inflammatory response in BDR rats.

Activated Protein C Attenuates Endotoxin-Induced Pulmonary Vascular Injury by Inhibiting Activated Leukocytes in Rats

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS-induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.