Number Of Infiltrating Leukocytes Research Articles

Selective Inhibition of Soluble Tumor Necrosis Factor Alters the Neuroinflammatory Response following Moderate Spinal Cord Injury in Mice.

Clinical and animal model studies have implicated inflammation and glial and peripheral immune cell responses in the pathophysiology of spinal cord injury (SCI). A key player in the inflammatory response after SCI is the pleiotropic cytokine tumor necrosis factor (TNF), which exists both in both a transmembrane (tmTNF) and a soluble (solTNF) form. In the present study, we extend our previous findings of a therapeutic effect of topically blocking solTNF signaling after SCI for three consecutive days on lesion size and functional outcome to study the effect on spatio-temporal changes in the inflammatory response after SCI in mice treated with the selective solTNF inhibitor XPro1595 and compared to saline-treated mice. We found that despite comparable TNF and TNF receptor levels between XPro1595- and saline-treated mice, XPro1595 transiently decreased pro-inflammatory interleukin (IL)-1β and IL-6 levels and increased pro-regenerative IL-10 levels in the acute phase after SCI. This was complemented by a decrease in the number of infiltrated leukocytes (macrophages and neutrophils) in the lesioned area of the spinal cord and an increase in the number of microglia in the peri-lesion area 14 days after SCI, followed by a decrease in microglial activation in the peri-lesion area 21 days after SCI. This translated into increased myelin preservation and improved functional outcomes in XPro1595-treated mice 35 days after SCI. Collectively, our data suggest that selective targeting of solTNF time-dependently modulates the neuroinflammatory response by favoring a pro-regenerative environment in the lesioned spinal cord, leading to improved functional outcomes.

Abstract WP143: Acute Treatment With Purinergic Receptor P2X4 Inhibitors Show Neuroprotective and Neuro-Rehabilitation Potential in Ischemic Stroke

Introduction: Stroke remains a leading cause of disability in the United States. Despite recent advances, interventions to reduce damage and enhance recovery after stroke is lacking. Therefore, validating new drug targets is critical for stroke treatment. P2X4, a receptor for adenosine triphosphate ATP, regulate activation of myeloid immune cells (both infiltrating monocytes/macrophages and brain-resident microglia) after stroke injury. Over-stimulation of P2X4Rs, due to excessive ATP release from the dying or damaged neuronal cells, contributes to ischemic injury. In this study, we pharmacologically targeted P2X4R to control immune response of myeloid cells during acute stroke phase and studied recovery at acute or chronic time point after stroke. Methods: We subjected 8-12 weeks old wild type mice of both sexes to 60 min right middle cerebral artery occlusion (MCAo) followed by 3 (acute) or 30 (chronic) days of reperfusion. We performed histological, behavioral (sensorimotor, anxiety and depressive), and flow cytometric analyses to determine the acute and chronic outcomes after P2X4R inhibitor 5-BDBD (1mg/kg P.O daily x3 days started after 4 hrs. of MCAo) treatment. Results: Treatment with 5-BDBD for 3 days not only significantly (*P<0.05) reduced infarct volume (40.94 ± 7.512 N=7 vs 21.21 ± 7.372 N=11, unpaired t-test) and neurological deficit (ND) score at 3 days post-stroke but also showed a progressive recovery on motor and balance coordination using rotarod test (*P<0.05) during chronic recovery at 30 days. 5-BDBD treatments also improved (*P<0.01) anxiety like behavior during the chronic recovery. We did not see any effect on depression like behavior. Flow cytometric analysis reveals that 5-BDBD treatment significantly reduced (*P<0.05) total number of infiltrated leukocytes. Subsequent analysis revealed that among those infiltrated leukocytes the number of Ly6C +hi pro-inflammatory monocytes was reduced in 5-BDBD vs. vehicle treated mice. Conclusions: Acute P2X4R inhibition protects against ischemic injury at both acute and chronic time point after stroke. Reduced number of infiltrated Ly6C +hi proinflammatory monocyte in 5-BDBD treated mice suggests its potential mechanism of neuroprotection after stroke.

Repetitive Exposure of IL-17 Into the Murine Air Pouch Favors the Recruitment of Inflammatory Monocytes and the Release of IL-16 and TREM-1 in the Inflammatory Fluids.

The infiltration of Th17 cells in tissues and organs during the development of many autoimmune diseases is considered a key step toward the establishment of chronic inflammation. Indeed, the localized and prolonged release of IL-17 in specific tissues has been associated with an increased severity of the inflammatory response that remains sustained over time. The cellular and molecular mechanisms behind these effects are far from being clear. In this study we investigated the effects of two repetitive administration of recombinant IL-17 into the murine air pouch to simulate a scenario where IL-17 is released over time in a pre-inflamed tissue. Consistent with our previous observations, mice receiving a single dose of IL-17 showed a transitory influx of neutrophils into the air pouch that peaked at 24 h and declined at 48 h. Conversely, mice receiving a double dose of the cytokine—one at time 0 and the second after 24 h—showed a more dramatic inflammatory response with almost 2-fold increase in the number of infiltrated leukocytes and significant higher levels of TNF-α and IL-6 in the inflammatory fluids. Further analysis of the exacerbated inflammatory response of double-injected IL-17 mice showed a unique cellular and biochemical profile with inflammatory monocytes as the second main population emigrating to the pouch and IL-16 and TREM-1 as the most upregulated cytokines found in the inflammatory fluids. Most interestingly, mice receiving a double injection of IL-1β did not show any change in the cellular or biochemical inflammatory response compared to those receiving a single injection or just vehicle. Collectively these results shed some light on the function of IL-17 as pro-inflammatory cytokine and provide possible novel ways to target therapeutically the pathogenic effects of IL-17 in autoimmune conditions.

Abstract 452: Serine Carboxypeptidase 1 Mediates Vascular Remodeling by Increasing Inflammatory Cell Infiltration

In pathological vascular remodeling, contractile vascular smooth muscle cells (VSMCs) switch their phenotype to highly proliferative and migratory states leading to neointimal formation. Inflammatory cell recruitment and infiltration, which is dependent on the increased expression of adhesion molecules on the endothelial cells, is a key event to initiate SMC phenotypic modulation in vascular remodeling. Serine carboxypeptidase 1 (scpep1), a novel protease containing the putative catalytic triad (Ser-Asp-His) common to all members of the serine protease family, has been proved to be involved in vascular remodeling by promoting SMC proliferation and migration in a catalytic triad-dependent manner. To determine whether Scpep1 modulates leukocyte adhesion and infiltration, a flow-induced model of vascular remodeling was conducted in wild-type (WT) or Scpep1 knockout (KO) mice. Scpep1-null mice show a decreased number of infiltrated leukocytes into the intima and media compared to WT mice. Further, mice devoid of Scpep1 show a dramatic reduction of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) expression in vessels in comparison with that of WT mice. Consistent with our in vivo data, the expression levels of ICAM-1 and VCAM-1 on human umbilical vein endothelial cells (HUVECs) transfected with SiRNA against Scpep1 were significantly decreased after TNF-α treatment. Taken together, these data suggest that Scpep1 may increase leukocyte extravasation by increasing the expression of VCAM-1 and ICAM-1 adhesion molecules.

Green tea extract supplementation ameliorates CCl4-induced hepatic oxidative stress, fibrosis, and acute-phase protein expression in rat

We evaluated the long-term effects of green tea extract (GTE) supplementation on oxidative stress, biliary acute phase protein expression, and liver function in CCl(4)-induced chronic liver injury. We evaluated the antioxidant activity of GTE in comparison with those of vitamin C, vitamin E, and β-carotene in vitro by using an ultrasensitive chemiluminescence analyzer. Chronic liver injury was induced by intraperitoneally administering carbon tetrachloride (CCl(4)) (1 mL/kg body weight, twice weekly) to female Wistar rats for 8 weeks. The effects of low (4 mg/kg body weight per day) and high (20 mg/kg body weight per day) doses of intragastric GTE on CCl(4)-induced liver dysfunction and fibrosis were examined by measuring the bile and blood reactive oxygen species levels and biochemical parameters by using Western blot and two-dimensional polyacrylamide gel electrophoresis techniques. GTE has greater scavenging activity against O(2)(-), H(2)O(2), and Hypochlorous acid (HOCl) in vitro than vitamin C, vitamin E, and β-carotene do. In vivo, CCl(4) markedly increased bile and blood reactive oxygen species production, lipid accumulation, number of infiltrated leukocytes, fibrosis, hepatic hydroxyproline content, and plasma alanine aminotransferase and aspartate aminotransferase activities, and reduced plasma albumin levels. Two-dimensional polyacrylamide gel electrophoresis revealed that CCl(4) increased the acute-phase expression of six biliary proteins and decreased hepatic B-cell lymphoma 2 (Bcl-2), catalase, and CuZn superoxide dismutase protein expression. GTE supplementation attenuated CCl(4)-enhanced oxidative stress, levels of biochemical parameters, pathology, and acute-phase protein secretion, and preserved antioxidant/antiapoptotic protein expression. GTE supplementation attenuates CCl(4)-induced hepatic oxidative stress, fibrosis, acute phase protein excretion, and hepatic dysfunction via the antioxidant and antiapoptotic defense mechanisms.

Split-Liver Procedure and Inflammatory Response: Improvement by Pharmacological Preconditioning

Final outcome of split-liver (SL) transplantation is impaired due to an increased rate of vascular complications and primary non-function. Herein, we hypothesized that an in situ split-liver procedure induces an inflammatory response and a deterioration of graft quality. We further studied whether graft quality can be improved by pharmacologic preconditioning. SL-procedure was performed in rats. One group (SL-HPP; n = 8) was pretreated according to a defined protocol [Homburg preconditioning protocol (HPP)], including pentoxyphylline, glycine, deferoxamine, N-acetylcysteine, erythropoietin, melatonin, and simvastatin. A second SL group (SL-Con; n = 8) received NaCl. Untreated non-SL served as controls (Sham; n = 8). Cytokines release, leukocyte invasion, endothelial activation and liver morphology were studied directly after liver harvest and after 8 h cold storage. Lung tissue was studied to determine remote injury. The SL-procedure induced an increase of TNF-α concentration, intercellular-adhesion-molecule 1 (ICAM-1) expression, leukocytic-tissue infiltration and vacuolization. This was associated with an increased number of apoptotic hepatocytes. HPP reduced TNF-α release, ICAM-1 expression, the number of infiltrated leukocytes, as well as hepatocellular vacuolization and apoptosis. In lung tissue, the SL-procedure caused an increased IL-1 and IL-6 concentration and leukocyte infiltration. HPP was capable of abrogating cytokine-mediated leukocytic response. Pharmacologic preconditioning of liver donors prevents the SL procedure-mediated inflammatory response, resulting in an improved graft quality.

Linoleic acid metabolite suppresses skin inflammation and tumor promotion in mice: possible roles of programmed cell death 4 induction

(+/-)-13-Hydroxy-10-oxo-trans-11-octadecenoic acid (13-HOA) is one of the lipoxygenase metabolites of linoleic acid (LA) from corn germ. Recently, we reported that this metabolite suppressed the expression of lipopolysaccharide-induced proinflammatory genes in murine macrophages by disrupting mitogen-activated protein kinases and Akt pathways. In this study, we investigated the inhibitory effects of 13-HOA on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in ears and skin, as well as tumor promotion in female ICR mice. Pretreatment with 13-HOA (1600 nmol) inhibited ear edema formation by 95% (P < 0.05) in an inflammation test and reduced tumor incidence and the number of tumors per mouse by 40 and 64% (P < 0.05 each), respectively, in a two-stage skin carcinogenesis model. Histological examinations revealed that it decreased epidermal thickness, the number of infiltrated leukocytes and cell proliferation index. Furthermore, 13-HOA (8-40 muM) suppressed TPA-induced anchorage-independent growth of JB6 mouse epidermal cells by 70-100%, whereas LA was virtually inactive. 13-HOA (40 muM) inhibited TPA-induced activator protein-1 transactivation but not extracellular signal-regulated kinase1/2 activation. Interestingly, 13-HOA (40 muM and 1600 nmol in JB6 cells and mouse skin, respectively) induced expression of programmed cell death 4 (Pdcd4), a novel tumor suppressor protein. To our knowledge, this is the first report of a food factor that is able to induce Pdcd4 expression. Collectively, our results indicate that 13-HOA may be a novel anti-inflammatory and antitumor chemopreventive agent with a unique mode of action.

A novel approach to measure the contribution of matrix metalloproteinase in the overall net proteolytic activity present in synovial fluids of patients with arthritis

Despite decades of research, only a very limited number of matrix metalloproteinase (MMP) inhibitors have been successful in clinical trials of arthritis. One of the central problems associated with this failure may be our inability to monitor the local activity of proteases in the joints since the integrity of the extracellular matrix results from an equilibrium between noncovalent, 1:1 stoichiometric binding of protease inhibitors to the catalytic site of the activated forms of the enzymes. In the present work, we have measured by flow cytometry the net proteolytic activity in synovial fluids (SF) collected from 95 patients with osteoarthritis and various forms of inflammatory arthritis, including rheumatoid arthritis, spondyloarthropathies, and chronic juvenile arthritis. We found that SF of patients with inflammatory arthritis had significantly higher levels of proteolytic activity than those of osteoarthritis patients. Moreover, the overall activity in inflammatory arthritis patients correlated positively with the number of infiltrated leukocytes and the serum level of C-reactive protein. No such correlations were found in osteoarthritis patients. Members of the MMP family contributed significantly to the proteolytic activity found in SF. Small-molecular-weight MMP inhibitors were indeed effective for inhibiting proteolytic activity in SF, but their effectiveness varied greatly among patients. Interestingly, the contribution of MMPs decreased in patients with very high proteolytic activity, and this was due both to a molar excess of tissue inhibitor of MMP-1 and to an increased contribution of other proteolytic enzymes. These results emphasize the diversity of the MMPs involved in arthritis and, from a clinical perspective, suggest an interesting alternative for testing the potential of new protease inhibitors for the treatment of arthritis.

Characterization of Methylated Bovine Serum Albumin-Induced Allergic Inflammation in Rats

An air pouch type allergic inflammation in rats was induced using an insoluble cationic protein, methylated bovine serum albumin (MeBSA), as an antigen. Changes in vascular permeability, local tissue edema, histamine contents in the pouch fluid, and number of infiltrated leukocytes and chemotactic activity in the pouch fluid were analyzed during an 8-hour period after injecting the antigen solution into the air pouch of the immunized and nonimmunized rats. Vascular permeability during the first 30-min interval in the immunized rats was higher than that in the nonimmunized rats, reflecting a higher histamine level in the pouch fluid. However, both the increase in vascular permeability and histamine level in the immunized rats in this period were much lower than those induced by a soluble, noncationic antigen, azobenzenearsonate-conjugated acetyl bovine serum albumin. In the MeBSA-induced allergic inflammation model, a second peak of vascular permeability was induced at 2 h, and local tissue edema formation became apparent at 2 h, reaching a plateau at 4 h. A prominent increase in leukocyte infiltration, especially neutrophils, into the pouch fluid was induced at 4 h in accordance with an increase in chemotactic activity in the pouch fluid. These observations indicate that the acute phase of MeBSA-induced allergic inflammation is characterized by a weak anaphylactic response and a prominent neutrophil infiltration.