Platelet endothelial cell adhesion molecule-1 in neutrophil emigration during acute bacterial pneumonia in mice and rats

Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31) is an adhesion molecule believed to mediate transendothelial migration of neutrophils and other leukocytes after CD11/CD18-mediated adhesion. Our study evaluated the role of PECAM-1 in neutrophil emigration across the pulmonary capillaries and the bronchial microvasculature using blocking anti-PECAM-1 antibodies in mice and rats. Neutrophil emigration was induced by Escherichia coli, a stimulus eliciting CD11/CD18-dependent emigration, or Streptococcus pneumoniae, a stimulus inducing CD11/CD18-independent emigration. Although anti-PECAM-1 antibodies partially inhibited glycogen-induced neutrophil emigration into the peritoneum, neutrophil emigration across either the pulmonary capillaries or the bronchial microvasculature in response to either E. coli or S. pneumoniae was not prevented when the function of PECAM-1 was inhibited in either mice or rats. There was also no increase in the number of intravascular neutrophils within the bronchial vessels after treatment with anti-PECAM-1 antibody. These studies indicate that either CD11/CD18-dependent or -independent adhesion pathways may lead to PECAM-1-independent transendothelial migration through the pulmonary or the bronchial endothelium.     

Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium

Myocardial damage due to reperfusion of ischemic tissue is caused primarily by infiltrating neutrophils. Although leukocyte beta2 integrins (CD18) play a critical role, significant neutrophil emigration persists when CD18 is neutralized or absent. This study examined the role of leukocyte beta1 integrin (alpha4) and its endothelial ligand VCAM-1 in CD18-independent neutrophil migration across cardiac endothelium. In a mouse model of myocardial ischemia and reperfusion, we show that compared with wild-type mice, neutrophil infiltration efficiency was reduced by 50% in CD18-null mice; in both types of mice, myocardial VCAM-1 staining increased after reperfusion. In wild-type mice, antibodies against CD18, ICAM-1 (an endothelial ligand for CD18), or VCAM-1 given 30 minutes before ischemia did not block neutrophil emigration at 3 hours reperfusion. Although anti-VCAM-1 attenuated neutrophil emigration by 90% in CD18-null mice, it did not diminish myocardial injury. To determine if CD18-independent neutrophil emigration was a tissue-specific response, we used isolated peripheral blood neutrophils from wild-type or CD18-null mice and showed neutrophil migration across lipopolysaccharide-activated cultured cardiac endothelium is CD18-independent, whereas migration across endothelium obtained from inferior vena cava is CD18-dependent. Consistent with our in vivo findings, migration of CD18-deficient neutrophils on cardiac endothelial monolayers is blocked by antibodies against alpha4 integrin or VCAM-1. We conclude tissue-specific differences in endothelial cells account, at least partially, for CD18-independent neutrophil infiltration in the heart.     

Neutrophil cytoskeletal rearrangements during capillary sequestration in bacterial pneumonia in rats

RATIONALE: Neutrophils accumulate in pulmonary capillaries during acute inflammation. Initial events in injury recognition and sequestration do not occur through selectin-mediated rolling. Cytoskeletal rearrangements, as assessed by submembrane F-actin rims, result in poorly deformable neutrophils that may not pass through capillaries. OBJECTIVE: To test the hypothesis that neutrophils sequestering during pneumonia contain F-actin rims and to determine the roles of CD11/CD18, L-selectin expression, and neutrophil-platelet adhesion in neutrophil sequestration. METHODS: Neutrophils were compared in blood obtained simultaneously from venous and arterial sites before and 4 h after instillation of Streptococcus pneumoniae or Escherichia coli in rats. MEASUREMENTS AND MAIN RESULTS: At 4 h of pneumonia, the number of neutrophils was greater in the venous blood entering the lungs than in the arterial blood leaving the lungs, indicating that neutrophil sequestration was occurring. More neutrophils entering the lungs contained F-actin rims than did neutrophils exiting, and the venous-arterial difference in F-actin-rimmed neutrophil counts completely accounted for sequestration. In E. coli pneumonia, in which neutrophil adhesion is mediated by CD11/CD18, CD18 blockade 15 min before blood samples were obtained did not prevent this sequestration of F-actin-rimmed neutrophils. Neutrophils expressing high or low levels of L-selectin or of neutrophils that bound platelets while circulating did not preferentially sequester. CONCLUSIONS: Neutrophils with cytoskeletal rearrangements preferentially sequester within the lungs during pneumonia, and this sequestration is not due to CD11/CD18-mediated adhesion, L-selectin expression, or platelet adhesion to neutrophils, suggesting that cytoskeletal rearrangements result in sequestration of neutrophils.     

Neutrophil and monocyte adherence to and migration across monolayers of cytokine-activated endothelial cells: the contribution of CD18, ELAM-1, and VLA-4.

Pretreatment of endothelial cells with cytokines enhances the adherence of leukocytes, a process that is mediated by surface proteins expressed on both cell types. A three-dimensional model system for the simultaneous determination of leukocyte adherence and migration was used to study the contribution of CD11/CD18, endothelial leukocyte-adhesion molecule-1 (ELAM-1) and VLA-4 in neutrophil and monocyte adherence to and migration through cytokine-activated endothelial cells. Pretreatment of endothelial cells for 4 hours with recombinant interleukin-1 beta (rIL-1 beta) was found to enhance neutrophil adherence and migration to a much greater extent than monocyte adherence and migration. Neutrophil adherence was almost completely prevented by the combined use of monoclonal antibodies (MoAbs) against ELAM-1 and CD18. Although ELAM-1 has been designated an endothelial cell-specific cytokine-inducible receptor for neutrophils, we observed that ENA2, an anti-ELAM-1 MoAb, significantly reduced monocyte adherence about 30%. MoAbs against VLA-4, the ligand of the cytokine-inducible receptor VCAM-1, did not affect monocyte adherence. However, the combined use of the MoAbs against CD18, ELAM-1, and VLA-4 had a very strong and additive inhibitory effect on rIL-1 beta-induced monocyte adherence. The anti-CD18 MoAb reduced both rIL-1 beta-induced neutrophil and monocyte migration far below the level of the unstimulated controls, whereas neither the anti-ELAM-1 nor the anti-VLA-4 MoAb significantly affected the process of migration. Our results indicate that neutrophils and monocytes initially adhere to cytokine-activated endothelial cells by CD18-independent and (to a lesser extent) by CD18-dependent mechanisms and subsequently change gears to a completely CD18-dependent migratory mechanism.     

CD11b limits bacterial outgrowth and dissemination during murine pneumococcal pneumonia

Expression of CD11b is enhanced on neutrophils recruited to the lungs during bacterial pneumonia. To determine the role that CD11b plays in pneumonia, CD11b gene-deficient (CD11b(-/-)) mice and normal wild-type (wt) mice were intranasally infected with Streptococcus pneumoniae. CD11b(-/-) mice had an enhanced outgrowth of pneumococci in the lungs and an increased dissemination of the infection, which could be reproduced by treatment of wt mice with an anti-CD11b antibody. This reduced resistance was associated with higher neutrophil counts in bronchoalveolar lavage fluid and lung tissue and an exaggerated lung inflammatory response. CD11b is important for an effective defense against S. pneumoniae pneumonia but not for recruitment of neutrophils.     

Differential roles for beta2 integrins in experimental autoimmune bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune disease associated with autoantibodies directed against the hemidesmosomal antigens anti-BP230 and anti-B180. Neonatal mice injected with rabbit anti-mouse BP180 (mBP10) IgG develop a BP-like disease. Complement, immune complexes, mast cells, and neutrophils play a key role in subepidermal blistering in this animal model. In this study we investigated the role of beta2 integrins in experimental BP. Wild-type (WT) mice pretreated with neutralizing antibody against CD11a (LFA-1), CD11b (Mac-1), CD11a plus CD11b, or CD18 alone failed to develop BP when injected with pathogenic anti-mBP180 IgG. This was associated with a significant reduction in neutrophil accumulation in neutralizing antibody-treated mice. Mac-1-deficient (Mac-1 knockout [KO]) mice were resistant to experimental BP despite normal complement deposition and mast cell and neutrophil degranulation. Neutrophil infiltration in Mac-1 KO mice was severely impaired at 24 hours. However, more neutrophils accumulated in the skin of Mac-1 KO mice compared with WT mice at early time points (2-4 hours), which was associated with an increase in their survival as determined by apoptosis markers. These data suggest that beta2 integrins play differential roles in experimental BP: LFA-1 is required for neutrophil recruitment, while Mac-1 mediates late neutrophil accumulation and apoptosis of infiltrating neutrophils.     

Alpha4beta1 integrin (VLA-4) blockade attenuates both early and late leukocyte recruitment and neointimal growth following carotid injury in apolipoprotein E (-/-) mice

BACKGROUND: The alpha(4)beta(1) integrin (VLA-4) supports rolling and firm adhesion of leukocytes to inflamed tissues via ligation of VCAM-1 or fibronectin expressed on the activated endothelium. We tested the hypothesis that VLA-4 mediates leukocyte recruitment and neointimal growth after arterial injury in the atherosclerosis-prone apolipoprotein E (ApoE)-deficient mouse. METHODS: ApoE (-/-) mice fed a Western diet underwent air desiccation injury, and the expression patterns of VLA-4 and VCAM-1 were determined by immunohistochemistry (IHC). To determine the effect of targeted VLA-4 blockade on leukocyte recruitment and neointimal growth, ApoE (-/-) mice received an intraperitoneal injection of a VLA-4 neutralizing monoclonal antibody (PS/2) at the time of injury alone or over a prolonged administration course. Additional mice received an isotype control antibody. RESULTS: IHC demonstrated a marked increase in VLA-4 expression 7 days following injury. Prolonged administration of PS/2 resulted in a 72% reduction (p < 0.02) in neointimal growth 28 days following injury. IHC revealed a marked 95% reduction in neutrophil recruitment at 7 days and a 48% reduction in macrophage recruitment 28 days following injury with prolonged PS/2 administration. CONCLUSIONS: Prolonged VLA-4 blockade reduces leukocyte recruitment and neointimal growth following air desiccation injury in ApoE (-/-) mice. These findings demonstrate an important role for VLA-4 in the response to arterial injury.     

Mechanisms of granulocytosis in the absence of CD18

Genetic deficiency in CD18 leads to disease characterized by myeloid hyperplasia, including profound granulocytosis and splenomegaly. Myeloid hyperplasia could directly result from the disruption of CD18 functions essential to granulopoiesis or basal leukocyte trafficking. Alternatively, myeloid hyperplasia could be reactive in nature, due to disruption of essential roles of CD18 in leukocyte responses to microbial challenge. To distinguish between these mechanisms, the hematopoietic systems of lethally irradiated wild-type (WT) mice were reconstituted with either WT fetal liver cells or CD18-deficient fetal liver cells, or an equal mixture of both types of cells. Granulocytosis and splenomegaly developed in mice that received CD18-deficient fetal liver cells. Splenomegaly was prevented and granulocytosis was inhibited by more than 95% in mice that had received both CD18-deficient and WT fetal liver cells, suggesting that myeloid hyperplasia was largely reactive in nature. Consistent with this postulate, the circulating life spans in the blood and the fraction of neutrophils that incorporated BrdU in the bone marrow were not increased for CD18-deficient neutrophils compared with the WT. However, these animals did develop mild granulocytosis compared with mice reconstituted with WT cells alone, and a higher percentage of CD18-deficient leukocytes were neutrophils compared with the WT leukocytes. These observations suggest that the granulocytosis observed in the absence of CD18 occurs through at least 2 mechanisms: one that is dramatically improved by the presence of WT cells, likely reactive in nature, and a second that is independent of the WT hematopoietic cells, involving an alteration in the lineage distribution of blood leukocytes.     

Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells.

The expression and function of a new cytokine-induced endothelial cell adhesion protein, vascular cell adhesion molecule-1 (VCAM-1), was characterized in vitro by using a monoclonal antibody, MoAb 4B9, which recognizes a functional epitope on this protein. As determined by enzyme-linked immunosorbent assay and radioimmunoprecipitation of metabolically labeled cells, VCAM-1 was minimally expressed on unstimulated human umbilical vein endothelium (HUVE), but was rapidly induced by recombinant human tumor necrosis factor-alpha (rhTNF-alpha), rh interleukin-1, and lipopolysaccharide. In contrast to intercellular adhesion molecule-1, VCAM-1 was not induced on dermal fibroblasts or arterial smooth muscle cells after stimulation with rhTNF, or on keratinocytes after stimulation with rh interferon-gamma. MoAb 4B9 significantly inhibited the adherence of peripheral blood lymphocytes (PBL) and lymphocytic cell lines, but not neutrophils, to rhTNF-activated HUVE. The inhibitory effect of MoAb 4B9 on PBL adherence to HUVE was additive to that produced by the CD18 MoAb 60.3. These results show that VCAM-1 mediates a CD18-independent pathway of peripheral blood lymphocyte adherence to cytokine-stimulated HUVE. We propose that lymphocyte binding to VCAM-1, induced on endothelium by cytokines, may be an important component of lymphocyte emigration at sites of inflammation or immune reaction.     

Increased sensitivity to the C-X-C chemokine CINC/gro in a model of chronic inflammation

OBJECTIVE: The C-C chemokine MCP-1 elicits significant neutrophil emigration in rats with chronic adjuvant-induced inflammation, but not in naive animals. We examined responses to the C-X-C chemokine CINC/gro to determine whether this class of chemokine elicits altered neutrophil responses during chronic inflammation. METHODS: CINC/gro was superfused over mesenteric venules of naive rats or animals with chronic adjuvant-induced vasculitis. Antibodies were used to characterize adhesive mechanisms. RESULTS: CINC/gro elicited leukocyte transendothelial migration in adjuvant-immunized rats at 100-fold lower concentrations than required to elicit transmigration in naive animals. In both groups, neutrophils constituted > 95% of the leukocytes recruited by CINC/gro. Using in vitro chemotaxis assays, neutrophils from control and adjuvant-immunized rats responded equally to CINC/gro, suggesting differences in migration were not related to neutrophil phenotype. Differences in adhesion molecule usage were noted in vivo. In control animals, CD18 antibodies blocked CINC/gro-induced neutrophil adhesion and emigration. In adjuvant-immunized animals, an alpha 4-integrin antibody reduced adhesion and emigration, while a CD18 antibody selectively inhibited emigration. CONCLUSIONS: This study demonstrates increased sensitivity to a C-X-C chemokine in a model of chronic inflammation, implicates the alpha 4-integrin in neutrophil adhesion, and demonstrates that CD18 mediates leukocyte transendothelial migration independent from firm adhesion.     

ICAM-2 mediates neutrophil transmigration in vivo: evidence for stimulus specificity and a role in PECAM-1-independent transmigration

ICAM-2 has been implicated in leukocyte transmigration in vitro, but there is little in vivo evidence to support this. To address this, neutrophil migration was investigated in ICAM-2-deficient mice (KO) and in wild-type (WT) mice treated with an anti-ICAM-2 blocking monoclonal antibody (mAb) (3C4). In a peritonitis model, IL-1beta-induced accumulation of neutrophils was significantly reduced in mice treated with 3C4 (51% inhibition) and in KO mice (41% inhibition). In contrast, TNF-alpha- or thioglycolate-induced responses were not suppressed in KO mice. Analysis of IL-1beta-induced leukocyte responses in cremasteric venules of KO animals by intravital microscopy indicated a defect in transmigration (44% inhibition) but not rolling or adhesion. As found before, TNF-alpha-induced leukocyte transmigration was unaltered in the KO mice. WT mice treated with the anti-ICAM-2 mAb also exhibited a selective reduction in leukocyte transmigration in response to IL-1beta while an anti-ICAM-1 mAb inhibited both leukocyte adhesion and transmigration. Interestingly, mAb 3C4 significantly suppressed IL-1beta-induced neutrophil transmigration in PE-CAM-1 KO animals in the peritonitis model but not in the cremaster muscle. The findings provide direct evidence for the involvement of ICAM-2 in neutrophil transmigration in vivo, though this role appears to be stimulus specific. Furthermore, ICAM-2 appears capable of mediating PECAM-1-independent leukocyte transmigration.     

Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1.

Adherence of human eosinophils to cytokine-stimulated endothelial cells, which was only partially due to CD18-dependent pathways, was also mediated by binding to endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1). Eosinophils bound specifically to both recombinant soluble ELAM-1 and recombinant soluble VCAM-1. Eosinophil binding to recombinant soluble VCAM-1 and to transfected CHO cells expressing VCAM-1 was inhibited with anti-VCAM-1 (4B9) and anti-very late activation antigen 4 (anti-VLA-4; HP1/2 or HP2/1) monoclonal antibodies. Eosinophils, but not neutrophils, expressed VLA-4 detected by cytofluorography. Eosinophil adherence to tumor necrosis factor alpha-stimulated human umbilical vein endothelial cells was partially blocked by monoclonal antibodies against ELAM-1 (BB11) and VCAM-1 (4B9) and against VLA-4 (HP2/1). Thus, while both eosinophils and neutrophils can bind to activated endothelial cells by adherence to ICAM-1 and ELAM-1, only eosinophils expressed VLA-4 and adhered to VCAM-1 on activated endothelial cells. Eosinophil adherence to VCAM-1 might provide a mechanism contributing to the selective recruitment of eosinophils into tissue sites of inflammation.     

Induction of acute pleural inflammation by Staphylococcus aureus. I. CD4+ T cells play a critical role in experimental empyema

Bacterial empyema is a frequent complication of pneumonia in patients with acquired immunodeficiency syndrome (AIDS). A model of Staphylococcus aureus empyema was developed that closely resembles bacterial empyema in patients infected with human immunodeficiency virus (HIV). Results show a compartmentalized chemokine response in bacterial empyema. The chemokine levels were higher in the pleural compartment than in the peripheral circulation. Polymorphonuclear leukocyte counts, murine GRO-alpha (KC), and macrophage inflammatory protein-2 levels were significantly (P<.001) lower in CD4+ knockout (CD4 KO) mice pleural fluid than in CD4+ wild-type (CD4 WT) mice. The CD4 KO mice had poorer bacterial clearance than CD4 WT mice. During S. aureus infection, interleukin-10 levels increased in the CD4 KO mice, whereas interferon-gamma levels were increased in CD4 WT mice. CD4+ T cell depletion results in a decreased pleural chemokine response, decreased neutrophil influx into pleural space, and impaired bacterial clearance in empyema.     

Characterization of hematopoietic progenitor mobilization in protease-deficient mice

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9-deficient mice, NE x CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE x CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12 alpha protein expression in the bone marrow of Ne x CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization.     

T-lymphocytes modulate the microvascular and inflammatory responses to intestinal ischemia-reperfusion

OBJECTIVE: The overall objective of this study was to define the contribution of T-lymphocytes to the microvascular and inflammatory responses of the intestine to ischemia/reperfusion (I/R). METHODS: The superior mesenteric artery of wild-type (WT) and SCID mice was occluded for 45 minutes, followed by 30 minutes or 6 hours of reperfusion. Intravital fluorescence microscopy was used to monitor the extravasation of FITC-labeled albumin or the adhesion of carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled T-lymphocytes in mucosal venules of the postischemic intestine. Tissue myeloperoxidase (MPO) was used to monitor neutrophil accumulation in the intestine of WT and SCID mice. RESULTS: Although the number of adherent T-cells was not increased above baseline at 1 hour after reperfusion, significant T-cell adhesion (both CD4(+) and CD8(+)) was noted at 6 hours of reperfusion. The latter response was prevented by pretreatment with a blocking antibody directed against MAdCAM-1, but not ICAM-1 or VCAM-1. A significant increase in MAdCAM-1 expression was noted in both lymphoid (Peyer's patch) and nonlymphoid regions of the postischemic small bowel. The early (30 minutes after reperfusion) albumin extravasation elicited by gut I/R in WT mice was reduced in SCID mice. Reconstitution of SCID mice with T-lymphocytes restored the albumin leakage response to WT levels. The increased intestinal MPO caused by I/R (6 hours of reperfusion) in WT mice was attenuated in SCID mice; with reconstitution of SCID mice with T-cells the MPO response was restored. CONCLUSIONS: These findings indicate that intestinal I/R is associated with the recruitment of CD4+ and CD8+ T-cells, which is mediated by endothelial MAdCAM-1. T-cells seem to modulate the recruitment of neutrophils that occurs hours after reperfusion as well as the increased albumin extravasation that occurs within minutes after reperfusion.     

An L-selectin ligand distinct from P-selectin glycoprotein ligand-1 is expressed on endothelial cells and promotes neutrophil rolling in inflammation

Neutrophils recruited from the blood are key players in the innate immune response. Selectins play critical roles in neutrophil recruitment by mediating their tethering and rolling in inflamed venules. While the roles of P- and E-selectin in this process are well established, the mechanisms of L-selectin-mediated neutrophil recruitment remain elusive. One proposal is that tethering is mediated by L-selectin on flowing neutrophils interacting with P-selectin glycoprotein ligand-1 (PSGL-1) on adherent neutrophils. To clarify whether L-selectin-mediated neutrophil recruitment depends entirely on PSGL-1, we examined the impact of L-selectin deficiency in mice with a PSGL-1-deficient background. L-selectin and PSGL-1 double-knockout mice exhibited a higher increase in their peripheral blood neutrophil count and a worse defect in neutrophil recruitment into the inflamed peritoneum than PSGL-1-deficient mice. Intravital microscopy of inflamed cremaster muscle venules showed that L-selectindeficiency or antibody blockade of L-selectin reduced the residual leukocyte rolling in PSGL-1-deficient mice. Flow cytometric analyses showed that the endothelial cells from the cremaster muscle bound L-selectin in a PSGL-1-independent manner. These results provide evidence for the existence of an L-selectin ligand distinct from PSGL-1 in inflammation and indicate that such a ligand is expressed on endothelial cells, promoting neutrophil rolling in vivo.     

A role for apoptosis in the control of neutrophil homeostasis in the circulation: insights from CD18-deficient mice

The control of neutrophil turnover in the circulation is a key event in homeostasis and inflammation. Using CD18- deficient (CD18(-/-)) mice that show a 19-fold increase of blood neutrophil counts when compared with wild-type animals (CD18(+/+)), we found that apoptosis of peripheral neutrophils was significantly reduced from 27.4% in the wild-type to 4.8% in CD18(-/-) mice within 4 hours after isolation as measured by analysis of DNA content. This was confirmed by detecting CD16 expression, nuclear morphology, and internucleosomal DNA degradation. In contrast, no difference in apoptosis was observed in neutrophils derived from the bone marrow. Neutrophilia and delayed neutrophil apoptosis were also present in CD18(-/-)/interleukin 6 (IL-6(-/-)) double knockout mice. Moreover, plasma of CD18(-/-) mice was not able to delay apoptosis of CD18(+/+) neutrophils and plasma of CD18(+/+) mice did not augment apoptosis of CD18(-/-) neutrophils. However, CD18(-/-) neutrophils revealed an up-regulation of the antiapoptotic gene bcl-X(l) and a down-regulation of the proapoptotic gene bax-alpha compared with CD18(+/+) neutrophils suggesting that this delayed apoptosis. Accordingly, down-regulation of Bax-alpha using antisense technique delayed apoptosis and prolonged neutrophil survival. The replacement of the hematopoietic system of CD18(+/+) mice by a 1:1 mixture of CD18(+/+) and CD18(-/-) hematopoietic cells abolished the delay of apoptosis in peripheral CD18(-/-) neutrophils and prevented neutrophilia. Altogether, this suggests that a delay of neutrophil apoptosis in CD18(-/-) mice causes an alteration of neutrophil homeostasis, which may induce the massive increase of peripheral neutrophil counts. Thus, apoptosis seems to be critically involved in the control of neutrophil turnover in the circulation.     

The role of T-lymphocytes in hypercholesterolemia-induced leukocyte-endothelial interactions

OBJECTIVE: Hypercholesterolemia promotes the adhesion of leukocytes to vascular endothelium in large and microscopic blood vessels. Lymphocytes that can modulate endothelial cell adhesion molecule expression have been implicated in the altered structure and function of large arterial vessels associated with chronic hypercholesterolemia. This study assesses the contribution of CD4(+) and CD8(+) T-cells to acute inflammatory responses observed in the microcirculation of hypercholesterolemic mice. METHODS: Intravital microscopy was used to quantify baseline leukocyte-endothelial cell interactions in cremasteric postcapillary venules of wild-type (WT) and severe combined immunodeficient (SCID) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. A group of SCID-HC mice received splenocytes from WT-HC mice (WT-->SCID). Separate WT-HC groups were depleted of neutrophils or CD4(+) and/or CD8(+) T-cells. RESULTS: WT-HC mice, compared with WT-ND, exhibited exaggerated leukocyte adherence and emigration. These leukocytes were predominantly granulocytes. These responses were absent in neutropenic WT-HC mice. SCID-HC mice also showed significantly less leukocyte adherence and emigration than WT-HC mice. Elevated leukocyte adherence and emigration were restored in WT-->SCID mice, despite a continued absence of circulating blood lymphocytes. Selective depletion of either CD4(+) or CD8(+) cell populations attenuated HC-induced leukocyte adhesion but not emigration. However, simultaneous depletion of both CD4(+) and CD8(+) cells attenuated both leukocyte adhesion and emigration to ND levels. DISCUSSION: These findings indicate that both CD4(+) and CD8(+) T-cells contribute to granulocyte adhesion and emigration elicited in postcapillary venules by hypercholesterolemia.     

The critical role of hematopoietic cells in lipopolysaccharide-induced airway inflammation

Rapid and selective recruitment of neutrophils into the airspace in response to LPS facilitates the clearance of bacterial pathogens. However, neutrophil infiltration can also participate in the development and progression of environmental airway disease. Previous data have revealed that Toll-like receptor 4 (tlr4) is required for neutrophil recruitment to the lung after either inhaled or systemically administrated LPS from Escherichia coli. Although many cell types express tlr4, endothelial cell expression of tlr4 is specifically required to sequester neutrophils in the lung in response to systemic endotoxin. To identify the cell types requiring trl4 expression for neutrophil recruitment after inhaled LPS, we generated chimeric mice separately expressing tlr4 on either hematopoietic cells or on structural lung cells. Neutrophil recruitment into the airspace was completely restored in tlr4-deficient mice receiving wild-type bone marrow. By contrast, wild-type animals receiving tlr4-deficient marrow had dramatically reduced neutrophil recruitment. Moreover, adoptive transfer of wild-type alveolar macrophages also restored the ability of tlr4-deficient recipient mice to recruit neutrophils to the lung. These data demonstrate the critical role of hematopoietic cells and alveolar macrophages in initiating LPS-induced neutrophil recruitment from the vascular space to the airspace.