PMN Infiltration Research Articles

Inactivation of CD11b in a mouse transgenic model protects against sepsis-induced lung PMN infiltration and vascular injury

To inactivate chronically the beta2-integrin CD11b (Mac-1), we made a transgenic model in mice in which we expressed the CD11b antagonist polypeptide neutrophil inhibitory factor (NIF). Using these mice, we determined the in vivo effects of CD11b inactivation on polymorphonuclear leukocyte (PMN) function and acute lung injury (ALI) induced by Escherichia coli septicemia. In wild-type PMNs, CD11b expression was induced within 1 h after E. coli challenge, whereas this response was significantly reduced in NIF(+/+) PMNs. Coimmunoprecipitation studies showed that NIF associated with CD11b in NIF(+/+) PMNs. To validate the effectiveness of CD11b blockade, we compared PMN function in NIF(+/+) and Mac-1-deficient (Mac-1(-/-)) mice. Adhesion of both Mac-1(-/-) and NIF(+/+) PMNs to endothelial cells in response to LPS was reduced in both types of PMNs and fully blocked only by the addition of anti-CD11a monoclonal antibody. This finding is indicative of intact CD11a function in the NIF(+/+) PMNs but the blockade of CD11b function. CD11b inactivation in NIF(+/+) mice interfered with lung PMN infiltration induced by E. coli and prevented the increase in lung microvessel permeability and edema formation, with most of the protection seen in the 1-h period after the E. coli. Thus our results demonstrate that CD11b plays a crucial role in mediating lung PMN sequestration and vascular injury in the early phase of gram-negative septicemia. The NIF(+/+) mouse model, in which CD11b is inactivated by binding to NIF, is a potentially useful model for in vivo assessment of the role of PMN CD11b in the mechanism of vascular inflammation.

Protein kinase C-ζ inhibition exerts cardioprotective effects in ischemia-reperfusion injury

Ischemia followed by reperfusion (I/R) in the presence of polymorphonuclear leukocytes (PMNs) results in marked cardiac contractile dysfunction. A cell-permeable PKC-zeta peptide inhibitor was used to test the hypothesis that PKC-zeta inhibition could attenuate PMN-induced cardiac contractile dysfunction by suppression of superoxide production from PMNs and increase nitric oxide (NO) release from vascular endothelium. The effects of the PKC-zeta peptide inhibitor were examined in isolated ischemic (20 min) and reperfused (45 min) rat hearts reperfused with PMNs. The PKC-zeta inhibitor (2.5 or 5 microM, n = 6) significantly attenuated PMN-induced cardiac dysfunction compared with I/R hearts (n = 6) receiving PMNs alone in left ventricular developed pressure (LVDP) and the maximal rate of LVDP (+dP/dt(max)) cardiac function indexes (P < 0.01), and these cardioprotective effects were blocked by the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (50 microM). Furthermore, the PKC-zeta inhibitor significantly increased endothelial NO release 47 +/- 2% (2.5 microM, P < 0.05) and 54 +/- 5% (5 microM, P < 0.01) over basal values from the rat aorta and significantly inhibited superoxide release from phorbol-12-myristate-13-acetate-stimulated rat PMNs by 33 +/- 12% (2.5 microM) and 40 +/- 8% (5 microM) (P < 0.01). The PKC-zeta inhibitor significantly attenuated PMN infiltration into the myocardium by 46-48 +/- 4% (P < 0.01) at 2.5 and 5 microM, respectively. In conclusion, these results suggest that the PKC-zeta peptide inhibitor attenuates PMN-induced post-I/R cardiac contractile dysfunction by increasing endothelial NO release and by inhibiting superoxide release from PMNs thereby attenuating PMN infiltration into I/R myocardium.

Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1

Erythropoietin (EPO) prevents the myocardial dysfunction induced by ischemia/reperfusion (I/R). Since I/R-induced myocardial dysfunction is associated with an acute inflammatory response, we assessed the anti-inflammatory properties of EPO using in vitro and in vivo models of I/R. Isolated cardiac myocytes were exposed to anoxia/reoxygenation (A/R; the in vitro counterpart to I/R). Hearts were challenged with I/R in situ. In vitro, A/R increased myocyte oxidant stress and converted the myocytes to a proinflammatory phenotype (these myocytes induced PMN transendothelial migration). Pretreatment of the myocytes with EPO prevented the A/R-induced proinflammatory effects. EPO increased myocyte (1) nuclear translocation of AP-1 (c-fos/c-jun), (2) eNOS, but not iNOS, protein expression, and (3) NO production. An AP-1 "decoy" oligonucleotide prevented the induction of eNOS by EPO and reversed the beneficial effect of EPO. An inhibitor of phosphatidylinostol 3 (PI3)-kinase prevented the nuclear translocation of AP-1 induced by EPO. In vivo, in wild type mice, I/R induced an increase in myocardial MPO activity (indicative of PMN infiltration); an effect prevented by pretreatment of the mice with EPO. This anti-inflammatory effect of EPO was not observed in cardiac specific c-fos(-/-) mice. Collectively, these findings indicate that EPO can ameliorate the myocardial inflammatory response in both in vitro and in vivo models of I/R. This beneficial effect of EPO is mediated by eNOS-derived NO via a PI3-kinase-dependent activation of AP-1.

Conditional Macrophage Ablation Demonstrates That Resident Macrophages Initiate Acute Peritoneal Inflammation

The role played by resident macrophages (Mphi) in the initiation of peritoneal inflammation is currently unclear. We have used a conditional Mphi ablation strategy to determine the role of resident peritoneal Mphi in the regulation of neutrophil (PMN) recruitment in experimental peritonitis. We developed a novel conditional Mphi ablation transgenic mouse (designated CD11bDTR) based upon CD11b promoter-mediated expression of the human diphtheria toxin (DT) receptor. The murine DT receptor binds DT poorly such that expression of the human receptor confers toxin sensitivity. Intraperitoneal injection of minute (nanogram) doses of DT results in rapid and marked ablation of F4/80-positive Mphi populations in the peritoneum as well as the kidney, and ovary. In experimental peritonitis, resident Mphi ablation resulted in a dramatic attenuation of PMN infiltration that was rescued by the adoptive transfer of resident nontransgenic Mphi. Attenuation of PMN infiltration was associated with diminished CXC chemokine production at 1 h. These studies indicate a key role for resident peritoneal Mphi in sensing perturbation to the peritoneal microenvironment and regulating PMN infiltration.

Antithrombin III pretreatment reduces neutrophil recruitment into the lung in a rat model of abdominal sepsis

Antithrombin III (AT III) is a serine protease inhibitor and the mechanism of its anti-inflammatory action is still not understood. In the present study, we aimed to investigate the anti-inflammatory action of AT III on lung injury in a rat model of sepsis. Three groups of animals were used in this controlled study: the sham-operated group (sham, n = 3) which only underwent a laparotomy; the control group (control, n = 7) which underwent cecal ligation and perforation (CLP); and the AT III-treated group (AT III, n = 6) which underwent CLP and received intravenous (i.v.) 250 U/kg AT III 30 min before induction of sepsis. Rats were killed 24 h after induction of sepsis by needle aspiration of the right ventricle after a sternotomy, and the lungs and trachea were removed en bloc under ether anesthesia. Pulmonary accumulation of polymorphonuclear leukocytes (PMN) was assessed by measuring lung tissue myeloperoxidase (MPO) activity. Lipid peroxidation in lung tissue was assessed by tissue thiobarbituric acid reactive substance (TBARS) levels. The plasma prostacyclin level was assessed by the plasma 6-keto prostaglandin F(1alpha)(6-keto-PGF(1alpha)) level, which is a stable derivative of prostacyclin. Histopathological changes in lung tissue were assessed by PMN count in the capillaries and alveolar spaces. The lung tissue TBARS level, MPO activity and PMN count in the control group were significantly higher than in the AT III group (P < 0.05). The change in plasma 6-keto-PGF(1alpha) level in the AT III group was insignificant compared with the control group (P = 0.15). AT III prevented pulmonary infiltration of PMN and subsequent injury by the endothelial release of prostacyclin in CLP-induced sepsis.

Leukocyte Migration through the Basement Membrane: Ultrastructural-Cytochemical Observation of the Human Fetal Membrane in Women with Chorioamnionitis-Related Preterm Delivery

Polymorphonuclear leukocytes (PMNs) infiltrating the chorioamnion is one causal agent of preterm delivery. Although the basement membrane (BM) has been proven to be a barrier against PMN migration in various tissues, there is a lack of data focusing on the relationship between PMN morphology and chorion laeve BM. Using peroxidase enzyme-cytochemistry, we morphologically studied PMN infiltration into the chorioamnion, especially the chorion laeve BM. The accumulated PMNs lay side by side just beneath the BM, while the cytoplasmic processes of some PMNs penetrated the BM. Peroxidase deposits appeared not only in the leukocyte granule but also on the phagosomes, indicating that PMNs at the site were activated. These features resembled those of activated PMNs migrating through a filter according to the gradient of chemo-attractant. The chorion laeve BM thus may be a barrier to PMN migration through the chorioamnion. Activated PMNs and BM may be involved in the pathophysiology of preterm delivery.

Intravenous immunoglobulin attenuates mesenteric ischemia–reperfusion injury

Intravenous immunoglobulin (IVIG) has been found useful in the treatment of various clinical entities and its effect has been associated with inhibition of complement-mediated tissue damage. The aim of this study was to determine the ability of IVIG to protect against mesenteric ischemia–reperfusion (IR)-induced local and remote injury. Rats received vehicle or IVIG (150–600 mg/kg) 5 min prior to sham operation or 30 min of superior mesenteric artery occlusion, followed by 5, 120, or 240 min of reperfusion. IVIG reduced IR-induced mucosal injury without altering IR-induced increases in PMN infiltration or LTB 4 generation. At 5 min post IR, the deposition of IgG and C3 in the lamina propria and surface epithelial cells was attenuated by IVIG. The increased capillary leak, evident at 240 min, was inhibited by IVIG and coincided with a reduction in C3 deposition in lung tissue. The beneficial effects of IVIG may be related to the ability to scavenge deleterious products.

IL-8 is a key chemokine regulating neutrophil recruitment in a new mouse model of Shigella-induced colitis.

The lack of a mouse model of acute rectocolitis mimicking human bacillary dysentery in the presence of invasive Shigella is a major handicap to study the pathogenesis of the disease and to develop a Shigella vaccine. The inability of the mouse intestinal mucosa to elicit an inflammatory infiltrate composed primarily of polymorphonuclear leukocytes (PMN) may be due to a defect in epithelial invasion, in the sensing of invading bacteria, or in the effector mechanisms that recruit the PMN infiltrate. We demonstrate that the BALB/cJ mouse colonic epithelium not only can be invaded by Shigella, but also elicits an inflammatory infiltrate that, however, lacks PMN. This observation points to a major defect of mice in effector mechanisms, particularly the lack of expression of the CXC chemokine, IL-8. Indeed, this work demonstrates that the delivery of recombinant human IL-8, together with Shigella infection of the colonic epithelial surface, causes an acute colitis characterized by a strong PMN infiltrate that, by all criteria, including transcription profiles of key mediators of the innate/inflammatory response and histopathological lesions, mimics bacillary dysentery. This is a major step forward in the development of a murine model of bacillary dysentery.

Pretreatment with EPO reduces the injury and dysfunction caused by ischemia/reperfusion in the mouse kidney in vivo

Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells and is known to be up-regulated during states of hypoxia. Here we investigate the effects of renal ischemia/reperfusion (I/R) on the degree of renal dysfunction and injury with recombinant human EPO in mice when given as either a 3-day pretreatment, or upon reperfusion of the kidney. Mice were treated with EPO (1000 IU/kg/day subcutaneously) for 3 days, or treated with EPO (1000 IU/kg subcutaneously) upon reperfusion, and subsequently subjected to bilateral renal artery occlusion (30 minutes) and reperfusion (24 hours). At the end of experiments, the following indicators and markers of renal injury and dysfunction were measured: plasma urea, creatinine, and aspartate aminotransferase (AST), tissue myeloperoxidase (MPO) activity [for polymorphonuclear leukocyte (PMN) infiltration], and tissue malonaldehyde (MDA) levels (for tissue lipid peroxidation). Kidneys were used for histologic evaluation of renal injury. EPO was able to significantly attenuate the renal dysfunction and injury associated with I/R, as well as the tissue injury. The increase in renal MPO activity and, hence, the degree of PMN infiltration were also significantly reduced in EPO-treated mice. In addition, lipid peroxidation as a result of renal I/R injury was also attenuated in EPO-treated mice. The protection afforded by the pretreatment regime of EPO was greater than that of administering EPO as a single bolus upon reperfusion. We propose that different mechanisms underlie the protective effects seen with EPO when given as either a daily pretreatment or as a single bolus, which need to be further investigated.

Impact of ischemia-reperfusion on joint synovium in rabbits

目的 探讨肢体缺血再灌注(IR)对关节滑膜的损伤及其机制.方法80只新西兰大白兔随机分成正常组、缺血组和再灌注组(缺血8h后再灌注);分别于缺血后4、6、8、10h,再灌注1、3、6、12、24、72h和1、2、4、8、12周.取滑膜组织测定丙二醛(MDA)、超氧化物歧化酶(SOD)及乳酸的含量,观察形态学变化并作滑膜组织血管标记和多形核细胞(PMN)计数.结果IR早期滑膜组织有不同程度的变性和坏死,在随后的修复过程中毛细血管出现严重的狭窄和闭塞;SOD在缺血阶段即明显降低,持续到IR后8周;IR组MDA显著高于对照侧、缺血组和正常组;自缺血4h～IR 2周,乳酸含量持续在高水平;PMN计数:IR组显著高于对照组.免疫组化标记显示IR 8～12周血管数较健侧减少(P<0.05).结论IR损伤早期以组织的变性坏死为主,之后患侧逐渐演变成慢性滑膜炎和创伤性关节炎.修复过程中微循环障碍及代谢产物长期滞留和PMN作用可能是影响其修复的重要因素。

The CD11/CD18 (beta2) integrins modulate neutrophil caspase activation and survival following TNF-alpha or endotoxin induced transendothelial migration.

Neutrophils (PMN) are short-lived cells but their survival is often prolonged in inflammation. The beta2 (CD11/CD18) integrins are involved in PMN migration into inflammation but their role in PMN survival is not well understood. We investigated the role of beta2 integrins in PMN caspase activation, a key enzyme cascade in apoptosis. After 20 h, caspase activation (Western blotting) was markedly decreased in PMN cultured on fibrinogen, a ligand for Mac-1 (CD11b/CD18), but not on fibronectin or albumin. In the presence of TNF-alpha or endotoxin (LPS), blockade of CD18 (beta2 chain) with mAb markedly increased caspase activation in PMN on fibrinogen. PMN which migrated through endothelium in vitro in response to TNF-alpha, LPS, IL-1alpha, IL-8 or C5a contained 58% fewer active caspase positive PMN after 20 h than non-migrated PMN remaining on the endothelium. When beta2 (CD18) integrin or lymphocyte function antigen (LFA)-1 (CD11a) plus Mac1 (CD11b) were blocked by mAb (intact or Fab'), the proportion of migrated PMN (but not of non-migrated PMN) with active caspases was significantly increased (2-4-fold) and this was associated with accelerated PMN apoptosis and death. Thus, engagement of ligands on extracellular matrix and endothelium by the beta2 integrins Mac-1 and LFA-1 plays a role in delaying apoptosis in PMN recruited in response to LPS and TNF-alpha. Inhibition of beta2 integrin function may not only inhibit PMN infiltration, but also accelerate PMN clearance from inflamed tissue.

Exogenous and Endogenous Nitric Oxide Donors Improve Post-Ischemic Tissue Oxygenation in Early Pancreatic Ischemia/Reperfusion Injury in the Rat

Introduction: In pancreatic ischemia/reperfusion (IR) injury (IRI) the role of nitric oxide (NO) is not completely understood. Using a rat model of normothermic in situ IRI, the effect of endogenous and exogenous NO donors on post-ischemic tissue oxygenation and tissue damage was investigated. Methods: IR was induced by 2-hour normothermic in situ ischemia of a pancreatic tail segment pedunculated on the splenic vessels with 2 h of reperfusion in an untreated, an L-arginine- and a sodium-nitroprusside-treated group (Wistar rats, n = 7/group). Animals without ischemia served as controls. Tissue oxygenation (pO_2ti) was monitored using a pO₂-sensitive Clark-type electrode. Histological investigation was performed following a semiquantitative score (edema, vacuolization, PMN infiltration, necrosis). Plasma lipase was another marker of organ damage. Results: The administration of L-arginine and sodium nitroprusside caused a significant amelioration of the decrease in pO_2ti after reperfusion compared to IR animals (p < 0.05). Histological damage was also reduced in the NO donor groups (p < 0.05). After reperfusion, plasma lipase in the L-arginine-treated animals was significantly lower compared to IR and sodium nitroprusside (p < 0.05). Conclusions: The administration of both endogenous and exogenous NO donors is protective in IRI of the rat pancreas which can be seen by an improvement in post-ischemic tissue oxygenation which indicates better nutritive tissue perfusion, amelioration of the histological tissue injury and, in L-arginine animals, lower lipase levels. NO donors could be useful in the prevention and reduction of the pancreatic IRI.

Reduction of renal ischemia-reperfusion injury in 5-lipoxygenase knockout mice and by the 5-lipoxygenase inhibitor zileuton.

The role of 5-lipoxygenase (5-LOX) in the pathophysiology of renal ischemia/reperfusion (I/R) injury is not known. Here we investigate the effects of 1) the 5-LOX inhibitor zileuton and 2) 5-LOX gene knockout (5-LOX(-/-)) mice on renal dysfunction and injury caused by I/R of the kidney in mice. Wild-type mice treated with zileuton (3 mg/kg i.v.) or 5-LOX(-/-) mice were subjected to bilateral renal artery occlusion (30 min) followed by reperfusion (24 h). Plasma urea, creatinine, and aspartate aminotransferase (AST) were measured as markers of renal dysfunction and reperfusion injury. Kidneys were used for histological evaluation of renal injury. Renal myeloperoxidase activity was measured and used as an indicator of polymorphonuclear leukocyte (PMN) infiltration and renal expression of intercellular adhesion molecule-1 (ICAM-1) was determined using immunohistochemistry. Administration of zileuton before I/R significantly reduced the degree of renal dysfunction (urea, creatinine) and injury (AST, histology). In addition, zileuton reduced the expression of ICAM-1 and the associated PMN infiltration caused by I/R of the mouse kidney. Compared with wild-type mice, the degree of renal dysfunction, injury, and inflammation caused by I/R in 5-LOX(-/-) mice was also significantly reduced, confirming the pathophysiological role of 5-LOX in the development of renal I/R injury. We propose that 1) endogenous 5-LOX metabolites enhance the degree of renal injury, dysfunction, and inflammation caused by I/R of the kidney by promoting the expression of adhesion molecules, and 2) inhibitors of 5-LOX may be useful in the treatment of conditions associated with I/R of the kidney.

Early T Cell Response to Allografts Occuring Prior to Alloantigen Priming Up-Regulates Innate-Mediated Inflammation and Graft Necrosis

The early inflammatory response within organ allografts is initiated by ischemia/reperfusion (I/R) and promotes subsequent alloantigen-primed T cell recruitment into and rejection of the graft. Polymorphonuclear leukocyte (PMN)-mediated tissue damage is a primary component of the early inflammation in allograft rejection. We sought to compare and elucidate the mechanism of early PMN infiltration into cardiac isografts and allografts. Despite identical production of PMN attractant chemokines, PMN infiltration following reperfusion into syngeneic and allogeneic grafts was not equivalent. PMN infiltration into isografts peaked at 9 to 12 hours post-transplant and quickly resolved. In contrast, PMN infiltration into allografts continued to elevated levels, peaking at 24 hours post-reperfusion. This amplified PMN infiltration into allografts did not resolve until 72 hours post-reperfusion and was accompanied by marked parenchymal necrosis. This early innate inflammatory response was regulated by IFN-gamma-producing CD8+ T cells present in the recipient before detectable alloantigen T cell priming. Co-culture with CD62L(low) CD8+ T cells, but not CD62L(high) CD8+ or CD62L(low) CD4+ T cells, harvested from naïve animals induced allogeneic endothelial cells to express IFN-gamma-dependent chemokines. These data demonstrate CD8+ T cell-mediated attack on the vascular endothelium of allografts within hours following organ reperfusion that amplifies innate immune-mediated intra-graft inflammation and necrosis.

Resuscitation affects microcirculatory polymorphonuclear leukocyte behavior after hemorrhagic shock: role of hypertonic saline and pentoxifylline.

We have previously shown that lung injury following fluid resuscitation either with hypertonic saline (HS) or lactated Ringer's (LR) plus pentoxifylline (PTX) attenuated acute lung injury when compared with LR resuscitation. The objective of the present study is to determine whether our previous observations are accompanied by changes in polymorphonu-clear leukocyte (PMN) behavior. To study this, PMN-endothelial cell interactions, microcirculatory blood flow, lung histology, lung PMN infiltration (MPO, Myeloperoxidase), and lung intra-cellular adhesion molecule-1 (ICAM-1) expression were assessed in a controlled hemorrhagic shock model followed by LR, HS, and LR+PTX resuscitation in rodents. Rats (240-300 g) were bled to a mean arterial pressure (MAP) of 35 mm Hg for 1 hr and then randomized into three groups: HS (7.5% NaCl, 4 ml/kg); LR (3x shed blood); and LR+PTX (25 mg/kg). Additionally, total shed blood was reinfused. A sham group underwent no shock and no treatment. The internal spermatic fascia was exteriorized and the microcirculation was observed by closed-circuit TV coupled to a microscope, 2 and 6 hrs after treatment. The number of leukocytes sticking to the venular endothelium was determined 2 hrs after fluid resuscitation. Microcirculatory blood flow was measured by an optical Doppler velocimeter. Lung histology and lung MPO immunostaining were assessed at 6 hrs, and lung ICAM-1 expression was determined by immunostaining at 2 hrs following fluid resuscitation. Two hours after treatment, HS (1.4 +/- 0.4), LR+PTX (1.7 +/- 0.3), and sham (0.4 +/- 0.2) groups presented significant reductions in leukocyte adherence (cells/100 microm venule length), compared with the LR group (4.0 +/- 0.9, P < 0.05). No differences were observed 6 hrs after treatment on leukocyte adherence and microcirculatory blood flow. ICAM-1 expression was significantly higher in LR-treated animals compared with the HS, LR+PTX, and sham groups (P < 0.01). PMN infiltration and overall lung injury were significantly attenuated by HS and LR+PTX. These results support earlier studies that indicated the potential application of HS and PTX in shock therapy and the increase in PMN-endothelial cell interaction and lung injury after LR resuscitation.

Paradoxical effects of elastase inhibitor guamerin on the tissue repair of two different wound models: sealed cutaneous and exposed tongue wounds.

Innate elastase inhibitors are known to be putatively involved in the regulation of tissue inflammation by inhibiting polymorphonuclear leukocyte (PMN) derived proteinases. The aim of this study was to evaluate affects of leukocyte elastase suppression and PMN infiltration on wound healing in mouse by administering the recombinant elastase inhibitor guamerin (rEIG) in two different wound models; 1) impaired pin-punctured dorsal mucosa of anterior tongue wound, 60 mice, treated with saline containing rEIG that were fed ad libitum and 2) stable linear excisional cutaneous wound, 40 mice, covered with fibrin sealant containing rEIG. The progress of healing was analyzed by histological methods. The tongue wounds treated with rEIG became edematous around the pin-punctured tongue wound, and influx of inflammatory cells and PMN into the underlying stromal tissue were seen rapidly after wounding and peaked between 2-4 days. Whereas the control mice showed almost no wheal formation in the pin-punctured wound, a far lesser levels of PMN infiltration, and almost complete wound closure in 4 days. In the other model, the liner excisional cutaneous wound treated with fibrin sealant containing rEIG showed early wound constriction, lesser degree of inflammatory cells influx, and complete reepithelialization in 4-5 days, whereas the wound of control mice with the fibrin sealant alone showed contrary delayed reepithelialization, greater degree of inflammatory cell infiltration, and consequencial formation of greater granulation tissue at wound site. Taken together, these data suggest paradoxical effects of rEIG on the wound healing where in the wound exposed to infiltrating milieu of microorganisms in the oral cavity, the rEIG aggravates the wound healing by interfering with other innate defensive factors and extended greater flux of PMNs to inflamed wound site, while in the wound enclosed by fibrin, the rEIG accelerated wound healing by inhibiting the inflammation-generated proteases and the acute inflammatory reaction.

Expression of L-histidine decarboxylase in granules of elicited mouse polymorphonuclear leukocytes.

Infiltrating polymorphonuclear leukocytes (PMN) in the peritoneal cavity were found to express L-histidine decarboxylase (HDC), the rate-limiting enzyme of histamine synthesis, in a csein-induced peritonitis model. Expression of HDC was detected in the elicited PMN, but not in the peripheral blood leukocytes. The peritoneal lavage fluids in this model were found to augment histamine synthesis in PMN isolated from the bone marrow. Rapid post-translational processing of HDC was observed in PMN, and the dominant form of HDC was the mature 53-kDa form, which was found to co-localize with a granule enzyme, matrix metalloproteinase-9 (MMP-9). Treatment of PMN with the phorbol ester PMA, which stimulates the release of MMP-9, did not liberate the granular HDC. Immunofluorescence studies using an anti-HDC antibody strongly suggested that HDC is bound to the cytosolic side of the granule membranes. These observations suggest that HDC is induced upon infiltration of PMN into the mouse peritoneal cavity and that histamine is synthesized by HDC attached to the granule membranes of PMN.

040 A Chronic Pressure Sore Nude Mouse Model

The epidemiology of pressure sores is not clearly understood yet. Tissue layers involved in the pressure sores include skin, subcutaneous fat, deep fascia, muscle, and bone. Widely used experimental models lead to the pressure sores in the skin and the underlying fatty tissue only. The aim of the present study was to develop a clinically relevant deep pressure sore model in nude mice. METHODS: Six weeks old Balb/c nude mice weighing 25–30 g were used. After introduction of anesthesia a steel disk (Ø 5 mm, 0.5 mm thickness) was implanted under the musculus gluteus max.. The wound was closed with 4/0 sutures. The full recovery was allowed for 10 days. Animals with an insufficient healing were excluded. The pressure was induced using a Neodymium magnet (1.29 Tesla) in conjunction with the disk in un‐anesthetized mouse periodically. A two hours compression was followed by one hour recovery for 4, 6 or 8 cycles, mimicking clinical setting. The controls underwent the same procedure, including the disk implantation but without placement of the magnet. RESULTS: The pressure sore depth was found to be cycle dependent – with the highest degree after 8 cycles. The histologic evaluations revealed signs of necrosis in the skin and subcutaneous fat after 4 and in the muscle only after 8 cycle of compression. PMN infiltration into the skin, subcutaneous fat, the deep fascia, and muscle was found in all groups. CONCLUSION: This model with clinically relevant magnitude and duration of pressure application leads to an injury in all tissue layers similar to the patient setting. This nude mouse model can be used to investigate the mechanism of pressure sores development and to study new therapeutic approaches (e.g. human cells) without the need of immunosuppression.

Endothelin-3 induced mesenteric vasoconstriction and PMN infiltration in the rat small intestine: role of endothelin receptors.

The objectives of this study were to characterize endothelin (ET)-3-induced alterations in intestinal hemodynamics and to evaluate whether ET-3 administration alters the tissue levels of polymorphonuclear leukocytes (PMNs) and modulates the epithelial barrier function of the small intestine. ET-3 (100 pmol/kg/min) was infused into the superior mesenteric artery (SMA) for 10 min, and tissue samples were obtained 30 min after terminating the infusion. SMA blood flow was significantly decreased throughout the experiment following ET-3 infusion. Pretreatment with bosentan (ET-A and ET-B receptor antagonist), ET-B receptor antagonist BQ-788 or ET-A receptor antagonist BQ-485 completely inhibited the ET-3-induced decrease in the SMA blood flow. Similar results were obtained from the resistance data, in which ET-3-induced increases in SMA resistance were significantly reduced by all ET receptor antagonists. ET-3 administration significantly elevated tissue MPO activity, blood-to-lumen clearance of (51)Cr-EDTA and caused a marked microscopic damage in the intestinal mucosa. ET-3-induced elevations in tissue PMN infiltration and mucosal damage were significantly inhibited by pretreatments with ET-A or ET-B receptor antagonists. Overall, our data indicate that ET-3 causes microscopic damage, PMN infiltration and mucosal dysfunction in the rat small intestine. In addition, ET-3-induced hemodynamic alterations as well as tissue PMN infiltration and mucosal damage are mediated by both ET-A and ET-B receptors.

Sulfur mustard primes phagocytosis and degranulation in human polymorphonuclear leukocytes

Sulfur mustard (2,2′-bis-chloroethyl-sulfide; SM) is a chemical warfare vesicant that causes debilitating skin lesions. Although a great deal of work has focused on the direct effects of SM exposure on the epithelium, it is unclear how much the inflammatory response, induced by exposure, contributes to lesion pathogenesis. Keratinocytes exposed to SM express a number of inflammatory mediators and elicit a cellular infiltrate consisting largely of polymorphonuclear leukocytes (PMN). PMN infiltration into SM lesions occurs as early as 30 min and peaks after several hours postexposure, and, despite the relatively short half-life of SM, PMN infiltrating a lesion could be exposed to micromolar concentrations of the agent. Previously, we have shown that exposure to low doses of sulfur mustard prime oxidative function in human PMN. The current study was undertaken to evaluate the effects of low-dose SM exposure on PMN phagocytosis, degranualtion and chemotaxis. PMN exposed to low doses of SM (50–200 μM) showed a dose-dependent enhancement of phagocytic function. Exocytosis of PMN azurophilic and specific granules [determined by analysis of granule-specific intravesicular receptors, Interleukin 10 receptor (IL-10R) and CD63] was also enhanced by SM exposure. Finally, we examined the effect of SM as a chemoattractant for PMN and show that SM is not itself a chemotaxin. These results suggest that SM injury may, in part, be caused by normal inflammatory function, and that therapeutic strategies aimed at down-regulating PMN activation could lessen the severity of SM injury and the time required for its resolution.