Despite having a high degree of sequence similarity, the Rho guanosine triphosphatases Rac1 and Rac2 regulate distinct functions in neutrophils. Here we demonstrate that the unique Rac2 localization and functions in neutrophils are regulated by two separate C-terminal motifs, the hypervariable domain and aspartic acid 150, one of which has not previously been linked to the function of Rho GTPases. In addition, we show an unexpected dependence of Rac1 localization on Rac2 activity in these same cells, demonstrating a degree of crosstalk between two closely related Rho GTPases. Thus, we have defined specific sequences in Rac that specify subcellular localization and determine the specificity of Rac2 in neutrophil chemotaxis and superoxide generation.
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A new rapid staining and measuring method has been developed for the quantification of migrated cells in a microchemotaxis chamber. The migrated cells were, after staining, evaluated by a transmission densitometer. The method introduced here is more accurate and faster than those described previously. In addition the technique can be used to determine the adherent capacity of cells.
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Background—Oxidized LDL has been found within the subendothelial space, and it exhibits numerous atherogenic properties, including induction of inflammatory genes. We examined the possibility that variations in endothelial response to minimally modified LDL (MM-LDL) constitute one of the genetic components in atherosclerosis.
Methods and Results—By a novel explant technique, endothelial cells (ECs) were isolated from the aorta of inbred mouse strains with different susceptibilities to diet-induced atherosclerosis. Responses to MM-LDL were evaluated by examining the expression of inflammatory genes involved in atherosclerosis, including monocyte chemotactic protein-1 (MCP-1) and macrophage-colony–stimulating factor (M-CSF), an oxidative stress gene, heme oxygenase-1 (HO-1), and other, noninflammatory, genes. ECs from the susceptible mouse strain C57BL/6J exhibited dramatic induction of MCP-1, M-CSF, and HO-1, whereas ECs from the resistant strain C3H/HeJ showed little or no induction. In contrast, ECs from the 2 strains responded similarly to lipopolysaccharide.
Conclusions—These data provide strong evidence that genetic factors in atherosclerosis act at the level of the vessel wall.
Chicken soup has long been regarded as a remedy for symptomatic upper respiratory tract infections. As it is likely that the clinical similarity of the diverse infectious processes that can result in “colds” is due to a shared inflammatory response, an effect of chicken soup in mitigating inflammation could account for its attested benefits. To evaluate this, a traditional chicken soup was tested for its ability to inhibit neutrophil migration using the standard Boyden blindwell chemotaxis chamber assay with zymosan-activated serum and fMet-Leu-Phe as chemoattractants. Chicken soup significantly inhibited neutrophil migration and did so in a concentration-dependent manner. The activity was present in a nonparticulate component of the chicken soup. All of the vegetables present in the soup and the chicken individually had inhibitory activity, although only the chicken lacked cytotoxic activity. Interestingly, the complete soup also lacked cytotoxic activity. Commercial soups varied greatly in their inhibitory activity. The present study, therefore, suggests that chicken soup may contain a number of substances with beneficial medicinal activity. A mild anti-inflammatory effect could be one mechanism by which the soup could result in the mitigation of symptomatic upper respiratory tract infections.
We evaluated the roles of the C-X-C chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) as well as the complement activation product C5a in development of lung injury after hindlimb ischemia-reperfusion in rats. During reperfusion, CD11b and CD18, but not CD11a, were upregulated on neutrophils [bronchoalveolar lavage (BAL) and blood] and lung macrophages. BAL levels of CINC and MIP-2 were increased during the ischemic and reperfusion periods. Treatment with either anti-CINC or anti-MIP-2 IgG significantly reduced lung vascular permeability and decreased lung myeloperoxidase content by 93 and 68%, respectively (P < 0.05). During the same period, there were significant increases in serum C5a-related neutrophil chemotactic activity. Treatment with anti-C5a decreased lung vascular permeability, lung myeloperoxidase, and BAL CINC by 51, 58, and 23%, respectively (P < 0.05). The data suggest that the C-X-C chemokines CINC and MIP-2 as well as the complement activation product C5a are required for lung neutrophil recruitment and full induction of lung injury after hindlimb ischemia-reperfusion in rats.
Neutrophil-mediated injury to gut epithelium may lead to disruption of the epithelial barrier function with consequent organ dysfunction, but the mechanisms of this are incompletely characterized. Because the epithelial apical junctional complex, comprised of tight and adherens junctions, is responsible in part for this barrier function, we investigated the effects of neutrophil transmigration on these structures. Using a colonic epithelial cell line, we observed that neutrophils migrating across cell monolayers formed clusters that were associated with focal epithelial cell loss and the creation of circular defects within the monolayer. The loss of epithelial cells was partly attributable to neutrophil-derived proteases, likely elastase, because it was prevented by elastase inhibitors. Spatially delimited disruption of epithelial junctional complexes with focal loss of E-cadherin, β-catenin, and zonula occludens 1 was observed adjacent to clusters of transmigrating neutrophils. During neutrophil transmigration, fragments of E-cadherin were released into the apical supernatant, and inhibitors of neutrophil elastase prevented this proteolytic degradation. Addition of purified leukocyte elastase also resulted in release of E-cadherin fragments, but only after opening of tight junctions. Taken together, these data demonstrate that neutrophil-derived proteases can mediate spatially delimited disruption of epithelial apical junctions during transmigration. These processes may contribute to epithelial loss and disruption of epithelial barrier function in inflammatory diseases.
Leukocyte recruitment in inflammation is critical for host defense, but excessive accumulation of inflammatory cells can lead to tissue damage. Neutrophil-derived serine proteases (cathepsin G [CG], neutrophil elastase [NE], and proteinase 3 [PR3]) are expressed specifically in mature neutrophils and are thought to play an important role in inflammation. To investigate the role of these proteases in inflammation, we generated a mouse deficient in dipeptidyl peptidase I (DPPI) and established that DPPI is required for the full activation of CG, NE, and PR3. Although DPPI–/– mice have normal in vitro neutrophil chemotaxis and in vivo neutrophil accumulation during sterile peritonitis, they are protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen. Specifically, there is no accumulation of neutrophils in the joints of DPPI–/– mice. This protective effect correlates with the inactivation of neutrophil-derived serine proteases, since NE–/– × CG–/– mice are equally resistant to arthritis induction by anti-collagen antibodies. In addition, protease-deficient mice have decreased response to zymosan- and immune complex–mediated inflammation in the subcutaneous air pouch. This defect is accompanied by a decrease in local production of TNF-α and IL-1β. These results implicate DPPI and polymorphonuclear neutrophil–derived serine proteases in the regulation of cytokine production at sites of inflammation.
Chronic obstructive pulmonary disease (COPD) is characterized by a neutrophilic airway inflammation that can be demonstrated by examination of induced sputum. Theophylline has antiinflammatory effects in asthma, and in the present study we investigated whether a similar effect occurs in COPD patients treated with low doses of theophylline. Twenty-five patients with COPD were treated with theophylline (plasma level of 9–11 mg/L) for 4 weeks in a placebo-controlled, randomized, double-blind crossover study. Theophylline was well tolerated. Induced sputum inflammatory cells, neutrophils, interleukin-8, myeloperoxidase, and lactoferrin were all significantly reduced by about 22% by theophylline. Neutrophils from subjects treated with theophylline showed reduced chemotaxis to N-formyl-met-leu-phe (∼ 28%) and interleukin-8 (∼ 60%). Neutrophils from a healthy donor showed reduced chemotaxis (∼ 30%) to induced sputum samples obtained during theophylline treatment. These results suggest that theophylline has antiinflammatory properties that may be useful in the long-term treatment of COPD.
The myeloproliferative disorders (MPD) are clonal diseases that originate from a transformed stem cell and involve all myeloid lineage. The affected cells have both proliferative and functional impairment. Therefore, we evaluated and compared neutrophil function in 31 patients with polycythemia vera (PV), idiopathic myelofibrosis (MF), chronic myeloid leukemia (CML), and essential thrombocytosis (ET). Neutrophil chemotaxis, random migration, bactericidal activity and superoxide anion release in these patients were simultaneously compared to those of 31 healthy controls. In this study, chemotactic activity was significantly impaired in patients with PV and CML as compared to controls (M+/-SE: 42 +/- 6 vs. 69+/- 5 cells/field; p<0.005 and 47+/-7 vs. 68+/- 5; p<0.05, respectively). The assessment of the bactericidal activity of neutrophils showed no impairment in most of the patients. In the CML group, the serum had a very strong “lytic” effect on bacteria, possibly due to the high levels of serum lysozyme (22 +/- 2 microgram/ml). The superoxide anion release was found to be normal in most of the patients. Nevertheless, in 25% of PV patients the superoxide production was impaired (less than 60% of the simultaneous controls). In ET most patients had normal neutrophil function. Regarding the effect of treatment, neutrophil chemotactic activity was found to be significantly reduced in the hydrea-treated patients, as compared to the non- treated patients (p<0.001) or healthy controls (<0.0001). We conclude that disturbances in neutrophil function are present in patients with various MPDs, except ET. This probably reflects abnormal maturation of ancessors of the damaged stem cells. Nevertheless, we should keep in mind that therapy itself could affect neutrophil functions. This matter should be studied more extensively. Although infections are not common in MPD disorders, they occasionally occur. It is possible that impairment in the phagocytic function contribute to the development of infections in patients with myeloproliferative disorders.
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