Early and advanced glycation end-products are increased in dietary copper deficiency

The hypothesis that nonenzymatic glycosylation of proteins (glycation) contributes to damage associated with dietary copper deficiency has depended largely on indirect evidence. Thus far, the observation of an elevated percentage of glycated hemoglobin in copper-deficient rats has provided the only direct evidence of an increase in glycation. We sought further direct evidence of increased glycation in copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient diet (CuD, 0.4 mg Cu/kg diet) for 5 weeks. Rats fed the CuD diet were copper deficient as judged by depressed organ copper concentrations and a variety of indirect indices. Measurements of hemoglobin A(1) and serum fructosamine (both early glycation end-products) as well as serum pentosidine (an advanced glycation end-product) indicated that all three compounds were elevated in CuD rats relative to CuA rats. This finding further supports the view that glycation is enhanced and thus may contribute to defects associated with dietary copper deficiency.     

Role of CD44 and hyaluronan in neutrophil recruitment

Lymphocyte CD44 interactions with hyaluronan localized on the endothelium have been demonstrated to mediate rolling and regulate lymphocyte entry into sites of chronic inflammation. Because neutrophils also express CD44, we investigated the role of CD44 and hyaluronan in the multistep process of neutrophil recruitment. CD44(-/-) and wild-type control mice were intrascrotally injected with the neutrophil-activating chemokine, MIP-2, and leukocyte kinetics in the cremasteric microcirculation were investigated 4 h subsequently using intravital microscopy. Neither the rolling flux nor the rolling velocities were decreased in CD44(-/-) mice relative to wild-type mice. In vitro, neutrophils did not roll on the CD44 ligand hyaluronan, consistent with the in vivo data that CD44/hyaluronan did not mediate rolling. However, the number of adherent leukocytes in the venule was decreased by 65% in CD44(-/-) mice compared with wild-type mice. Leukocyte emigration was also greatly decreased in the CD44(-/-) mice. The same decrease in adhesion and emigration was observed in the wild-type mice given hyaluronidase. Histology revealed neutrophils as being the dominant infiltrating population. We generated chimeric mice that express CD44 either on their leukocytes or on their endothelium and found that CD44 on both the endothelium and neutrophils was important for optimal leukocyte recruitment into tissues. Of those neutrophils that emigrated in wild-type and CD44(-/-) mice, there was no impairment in migration through the interstitium. This study suggests that CD44 can mediate some neutrophil adhesion and emigration, but does not appear to affect subsequent migration within tissues.     

Impaired deformability of copper-deficient neutrophils

We have previously shown that dietary copper deficiency augments neutrophil accumulation in the lung microvasculature. The current study was designed to determine whether a diet deficient in copper promotes neutrophil chemoattraction within the lung vasculature or if it alters the mechanical properties of the neutrophil, thus restricting passage through the microvessels. Sprague-Dawley rats were fed purified diets that were either copper adequate (6.3 microg Cu/g diet) or copper deficient (0.3 microg Cu/g diet) for 4 weeks. To assess neutrophil chemoattraction, bronchoalveolar lavage fluid was assayed for the neutrophil chemokine macrophage inflammatory protein-2 (MIP-2) by enzyme-linked immunosorbent assay. Neutrophil deformability was determined by measuring the pressure required to pass isolated neutrophils through a 5-microm polycarbonate filter. The MIP-2 concentration was not significantly different between the dietary groups (Cu adequate, 435.4 +/- 11.9 pg/ml; Cu deficient, 425.6 +/- 14.8 pg/ml). However, compared with controls, more pressure was needed to push Cu-deficient neutrophils through the filter (Cu adequate, 0.150 +/- 0.032 mm Hg/sec; Cu deficient, 0.284 +/- 0.037 mm Hg/sec). Staining of the filamentous actin (F-actin) with FITC-Phalloidin showed greater F-actin polymerization and shape change in the Cu-deficient group. These results suggest that dietary copper deficiency reduces the deformability of neutrophils by promoting F-actin polymerization. Because most neutrophils must deform during passage from arterioles to venules in the lungs, we propose that copper-deficient neutrophils accumulate in the lung because they are less deformable.     

Effect of copper deficiency on the lymphatic absorption of cholesterol, plasma chylomicron clearance, and postheparin lipase activities

The lymphatic absorption of cholesterol and plasma clearance of chylomicrons were investigated in Cu-deficient rats (CuD) fed 0.5 mg Cu/kg diet, as compared with Cu-adequate control rats (CuA) fed 7.5 mg/kg diet. Cholesterol absorption was measured by the 14C-radioactivity appearing in the mesenteric lymph at hourly intervals for 8 hr after an intraduodenal dose of [14C]cholesterol. The plasma clearance of chylomicrons was measured at 3, 6, and 10 min after an intravenous dose of chylomicrons labeled in vivo with [3H]retinyl ester. Cumulative [14C]cholesterol absorption and total lymphatic output of cholesterol were significantly decreased in CuD at 4 hr and thereafter, with no change in percentage distribution of free and esterified cholesterol. Over an 8-hr period, 7.3% of the dose was absorbed by CuD and 9.2% by CuA. When [3H]chylomicrons, obtained from a CuD or CuA donor rat, were injected into CuD and CuA recipient rats, the label was cleared faster in CuD during the first 3 min. At 6 and 10 min, however, no significant difference in percentage clearance of the dose was observed between the groups. The half-life (t1/2) of [3H]chylomicrons and the total 3H-radioactivity taken up by the liver during the entire 10-min period did not differ between the groups, regardless of the source of chylomicrons. The activities of both endothelial lipoprotein lipase (LPL) and hepatic lipase (HL) in postheparin plasma were markedly lower in CuD. As expressed in micromoles fatty acid released/hr/ml plasma, the activities of LPL in CuD and CuA were 32.6 +/- 1.9 and 45.6 +/- 1.3, respectively. A similar magnitude of difference was also observed in HL activity. The data provide evidence that copper deficiency impairs the intestinal transport of cholesterol and the peripheral lipolysis of chylomicrons. The data, however, strongly suggest that the hepatic uptake of chylomicron remnants via the apo-E-dependent mechanism may not be impaired in Cu deficiency.     

Neointima formation in the rat carotid artery is exacerbated by dietary copper deficiency

Dietary copper is an essential trace element with roles in both functional and structural aspects of the cardiovascular system. In particular, the vascular response to inflammatory stimuli is known to be significantly augmented in copper-deficient rats. The current study was designed to quantify the extent of injury-induced neointimal proliferation and stenosis in rats fed diets either adequate or deficient in copper. Male, weanling Sprague-Dawley rats were fed purified diets that were either adequate (CuA; 5.6 microg Cu/g) or deficient (CuD; 0.3 microg Cu/g) in copper for 4 weeks. Balloon injury was induced in the left external carotid arteries. Fourteen days after injury, histomorphometric analysis of cross-sections from carotid arteries showed increased neointimal formation in the CuD group compared with the CuA controls (neointima/media ratio: 4.55 +/- 0.93 vs 1.45 +/- 0.2, respectively). These results correspond with data indicating that the activity of Cu/Zn-superoxide dismutase (SOD) is depressed in rats fed this CuD diet. Because superoxide anion and redox status are known to play a key role in the extent of neointimal formation in response to injury, we propose that the exaggerated neointimal proliferation seen in the CuD group is the result of the diminished Cu/Zn-SOD activity.     

Signs of iron deficiency in copper-deficient rats are not affected by iron supplements administered by diet or by injection

The goal of this study was to determine the effects of Fe supplementation on the anemia of Cu deficiency in rats. In addition, we observed changes in serum and organ Cu and Fe during the development of Cu deficiency. In Experiment 1, weanling male Sprague-Dawley rats were fed AIN-93G diets containing either <0.3 mg Cu [Cu deficient (CuD)] or 6.0 mg Cu [Cu adequate (CuA)] per kilogram diet, and 35 mg Fe/kg. Five rats from each group were killed at intervals for the analysis of hematologic parameters and mineral content of various organs. In Experiment 2, two groups of 24 rats each were fed either the CuA diet or the CuD diet for 14 days. Then, three sets of eight rats in each group received three separate Fe treatments: (1) daily intraperitoneal injections of 400 mug Fe (Cu-free ferric citrate) per rat for another 14 days, (2) fed similar diets that contained three times the normal amount of Fe (105 mg/kg) for 14 days, or (3) received no further Fe treatment. At day 21, all rats were fed a 1-g meal labeled with (59)Fe to determine Fe absorption. After 28 days, rats were killed for the analyses of Fe and Cu status. Results of Experiment 1 showed that within 14 days, CuD rats had lower blood hemoglobin (Hgb), red blood cell count, and mean corpuscular volume than CuA rats. Copper concentrations in all tissues measured were lower in the CuD rats than in controls. Serum ceruloplasmin (Cp) activity in CuD rats was only 0.8% of CuA rats at day 7. During this period, enterocyte and liver Fe concentrations were elevated and serum Fe was reduced, but there was no change in spleen Fe. Results of Experiment 2 showed that CuD rats absorbed less Fe than CuA rats. Supplemental Fe by diet or by intraperitoneal injections did not prevent anemia in the CuD rats or affect other parameters of Cu status. Serum total iron binding capacity [transferrin (Tf)] was not changed by Cu deficiency or by Fe supplementation; however, percent Tf saturation was reduced in CuD rats but was not enhanced by Fe supplementation. These data suggest that anemia of Cu deficiency occurs because of reduced Fe absorption, and it inhibits release of Fe from the liver and inefficient loading of Fe into Tf because of very low plasma Cp activity. The latter then leads to inefficient delivery of Fe to the erythroid cells for heme and Hgb synthesis.     

Repletion of copper-deficient rats with dietary copper restores duodenal hephaestin protein and iron absorption

Copper (Cu) deficiency in rats reduces the relative concentration of duodenal hephaestin (Hp), reduces iron (Fe) absorption, and causes anemia. An experiment was conducted to determine whether these effects could be reversed by dietary Cu repletion. Five groups of eight weanling male rats each were used. Group 1 was fed a Cu-adequate diet (5.0 mg Cu/kg; CuA) and Group 2 was fed a Cu-deficient diet (0.25 mg Cu/kg; CuD) for 28 days. The rats were fed 1.0 g each of their respective diets labeled with 59Fe (37 kBq/g), and the amount of label retained was measured one week later by whole-body-counting (WBC). Group 3 was fed a CuA diet and Groups 4 and 5 were fed a CuD diet for 28 days. Group 5 was then fed the CuA diet for another week while Groups 3 and 4 continued on their previous regimens. Rats in Groups 3, 4, and 5 were fed 1.0 g of diet labeled with 59Fe, and the amount of label retained was measured by WBC one week later. Rats were killed and duodenal enterocytes isolated for Hp protein analysis, whole blood was analyzed for hematological parameters, and various organs for 59Fe content. CuD rats absorbed less (P<0.05) Fe than CuA rats, the relative amount of duodenal Hp was less (P<0.05) in CuD rats, and the CuD rats developed anemia. After the CuD rats had been repleted with Cu for one week, Fe retention rose to values even higher (P<0.05) than those in CuA rats. After two weeks, the relative amount of duodenal Hp was higher (P<0.05) than normal, and most signs of anemia were reversed. Liver 59Fe was elevated in CuD rats, but was restored to normal upon Cu repletion. These findings suggest a strong association between duodenal Hp abundance and Fe absorption in the CuD rat, and that reduced Fe absorption is an important factor in the cause of anemia.     

The lectin-like domain of complement receptor 3 protects endothelial barrier function from activated neutrophils

The adhesion of neutrophils to endothelial cells is a central event leading to diapedesis and involves the binding of the I-domain of beta(2) integrins (CD11/CD18) to endothelial ICAMs. In addition to the I-domain, the beta(2) integrin complement receptor 3 (CR3) (CD11b/CD18) contains a lectin-like domain (LLD) that can alter leukocyte functions such as chemotaxis and cytotoxicity. The present study demonstrates that, in contrast to the CR3 I-domain, Ab blockade of the CR3 LLD has no role in mediating neutrophil-induced loss of endothelial barrier function. However, activation of CR3 with the LLD agonist beta-glucan protects the barrier function of endothelial cells in the presence of activated neutrophils and reduces transendothelial migration without affecting adhesion of the neutrophils to the endothelium. The LLD site-specific mAb VIM12 obviates beta-glucan protection while activation of the LLD by VIM12 cross-linking mimics the beta-glucan response by both preserving endothelial barrier function and reducing neutrophil transendothelial migration. beta-glucan has no direct effect on endothelial cell function in the absence of activated neutrophils. These findings demonstrate that signaling through the CR3 LLD prevents neutrophil-induced loss of endothelial barrier function and reduces diapedesis. This suggests that the LLD may be a suitable target for oligosaccharide-based anti-inflammatory therapeutics.     

Migration of CD18-deficient neutrophils in vitro: evidence for a CD18-independent pathway induced by IL-8

Neutrophils isolated from a child with severe leukocyte adhesion deficiency 1 (LAD1) had a complete absence of expression of the CD11/CD18 beta2 integrin family of adhesion molecules, and were shown to be deficient in the in vitro adhesion and migration properties. However, we found that interleukin-8 (IL8), a potent chemoattractant for neutrophils, and sputum sol phase induced these LAD1 neutrophils to migrate through an endothelial cell layer in vitro, and confirmed that this migration was CD18-independent. These findings add to evidence of CD18-independent mechanisms of neutrophil recruitment, in particular neutrophil infiltration into the lungs, where IL8 may be an important recruitment factor.     

Mediators and regulation of neutrophil accumulation in inflammatory responses in lung: insights from the IgG immune complex model

Neutrophil trafficking in lung involves transendothelial migration, migration in tissue interstitium, and transepithelial migration. In a rat model of IgG immune complex-induced lung injury, it was demonstrated that neutrophil emigration involves regulatory mechanisms including complement activation, cytokine regulation, chemokine production, activation of adhesion molecules, and their respective counter receptors. The process is presumably initiated and modulated by the production of early response cytokines and chemokines from lung cells, especially from alveolar macrophages. TNF-alpha and IL-1 up-regulate intracellular adhesion molecule-1 (ICAM-1) and E-selectin, setting the stage for neutrophil migration through endothelium. The CXC chemokines, such as macrophage inflammatory protein (MIP)-2 and cytokine-inducible neutrophil chemoattractant (CINC), constitute chemokine gradient to orchestrate neutrophil migration in lung. Complement activation induced by IgG immune complex deposition is another important event leading to neutrophil accumulation in lung. Complement activation product C5a not only plays an important role in chemoattracting neutrophils into lung, but regulates adhesion molecules, chemokines, and cytokines expression. In addition, oxidative stress may regulate neutrophil accumulation in lung by modulation of adhesion molecule activation and chemokine production. In this review, we focus on the current knowledge of the mechanisms leading to accumulation of neutrophils during acute lung injury.     

Role of galectin-3 as an adhesion molecule for neutrophil extravasation during streptococcal pneumonia

Recruitment of neutrophils from blood vessels to sites of infection represents one of the most important elements of innate immunity. Movement of neutrophils across blood vessel walls to the site of infection first requires that the migrating cells firmly attach to the endothelial wall. Generally, neutrophil extravasation is mediated at least in part by two classes of adhesion molecules, beta(2) integrins and selectins. However, in the case of streptococcal pneumonia, recent studies have revealed that a significant proportion of neutrophil diapedesis is not mediated by the beta(2) integrin/selectin paradigm. Galectin-3 is a beta-galactoside-binding lectin implicated in inflammatory responses as well as in cell adhesion. Using an in vivo streptococcal pneumonia mouse model, we found that accumulation of galectin-3 in the alveolar space of streptococcus-infected lungs correlates closely with the onset of neutrophil extravasation. Furthermore, immunohistological analysis of infected lung tissue revealed the presence of galectin-3 in the lung tissue areas composed of epithelial and endothelial cell layers as well as of interstitial spaces. In vitro, galectin-3 was able to promote neutrophil adhesion to endothelial cells. Promotion of neutrophil adhesion by galectin-3 appeared to result from direct cross-linking of neutrophils to the endothelium and was dependent on galectin-3 oligomerization. Together, these results suggest that galectin-3 acts as an adhesion molecule that can mediate neutrophil adhesion to endothelial cells. However, accumulation of galectin-3 in lung was not observed during neutrophil emigration into alveoli induced by Escherichia coli infection, where the majority of neutrophil emigration is known to be beta(2) integrin dependent. Thus, based on our results, we propose that galectin-3 plays a role in beta(2) integrin-independent neutrophil extravasation, which occurs during alveolar infection with Streptococcus pneumoniae.     

Hyperbaric hyperoxia exaggerates respiratory membrane defects in the copper-deficient rat lung

Scanning (SEM) and transmission electron microscopy (TEM) were used to examine the effect of dietary copper deficiency and hyperbaric hyperoxia, alone and in combination, on lung structure. Male, weanling Sprague-Dawley^† rats were fed a copper-deficient (CuD, 0.2 μg/g) or copper-adequate diet (CuA, 5.1 μg/g). After 35–41 d on their respective diets, rats from each group were placed inside a pressure vessel kept at 27°C under one of two pressure protcols. Air controls were maintained at 1 atm for 75 min. Rats exposed to oxygen were maintained at 1 atm of air plus 3 atm of oxygen for 1 h and then decompressed for 15 min. Under SEM, none of the treated lungs (CuD, CuA-O₂ exposed, or CuD-O₂ exposed) showed abnormal lung morphology from the conducting bronchioles down to the alveoli. Copper-deficient red blood cells were abnormally shaped. Under TEM, CuA-O₂-exposed lungs showed thicker respiratory membranes, especially basement membranes and endothelial cells, and alveolar Type II cells having more than the usual number of surfactant vacuoles. CuD lungs also showed thicker endothelial and basement membrane components of the respiratory membrane, but normal looking Type II cells. CuD-O₂-exposed lungs showed greatly thickened respiratory membranes and severe disruption of both endothelium and basement membrane and, judging by the increased number of nuclei per field, an increase in the number of both Type I and Type II cells. We conclude that copper deficiency enhances the damage caused by O₂ toxicity, an effect that may be caused by reduced antioxidant status.     

The effect of anti-intercellular adhesion molecule-1 on phorbol-ester-induced rabbit lung inflammation

The role of the CD18 complex of leukocyte glycoproteins in adhesion-dependent functions of human leukocytes in vitro has been well documented. A ligand, intercellular adhesion molecule-1 (ICAM-1), for at least one member of the CD18 complex has been identified. This molecule is inducible on many cell types including vascular endothelium and keratinocytes by inflammatory mediators such as IL-1, TNF, and IFN-gamma. ICAM-1 has been shown to mediate, in part, the in vitro adhesion of lymphocytes and neutrophils to endothelial cells expressing ICAM-1. In the present study we have shown that mAb's to the human CD18 complex and to human ICAM-1 cross react with rabbit cells and that both anti-CD18 and anti-CD11b but neither anti-CD11a nor anti-ICAM-1 mAb's inhibit neutrophil migration, an adhesion-dependent function, in vitro. Pretreatment of rabbits with anti-CD18 and anti-ICAM-1 but not anti-CD11a mAb inhibited by greater than 60% neutrophil migration into PMA-induced inflamed rabbit lungs. This effect of anti-ICAM-1 mAb on pulmonary neutrophil influx after PMA injection has important implications. Specifically, that ICAM-1 can function as a ligand for CD18 and can mediate, at least in part, the migration of neutrophils to inflammatory sites.     

The strength of integrin binding between neutrophils and endothelial cells

The firm adhesion of activated polymorphonuclear neutrophils to endothelial cells in blood vessels is achieved through binding of the integrin intercellular adhesion molecule. To contribute to the better understanding of this adhesion step, our investigation is aimed at the relationship between integrin expression and the strength of neutrophil binding to endothelial cells. Flow cytometry and 3D scanning microscopy are used to study integrin expression and distribution, respectively. It is found that CD11b/CD18 integrin expression is localized in clusters distributed irregularly over the neutrophil surface. After cell activation, the cluster distribution polarizes, increasing the local CD11b/CD18 density concurrently with nearly doubled integrin expression. The neutrophil adhesion efficiency is measured in a flow chamber coated successively by various substrates, including endothelial cells in an activated state. Analysis of the flow dependence of the number of attached cells reveals the prevailing number of neutrophils with stronger binding to the endothelium when both cells are in the activated state in comparison with non-activated cells.     

Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion.

The human lymphocyte homing receptor, LAM-1, mediates the adhesion of lymphocytes to specialized high endothelial venules (HEV) of peripheral lymph nodes. We now report that LAM-1 is also a major mediator of leukocyte attachment to activated human endothelium. In a novel adhesion assay, LAM-1 was shown to mediate approximately 50% of the adhesion of both lymphocytes and neutrophils to TNF-activated human umbilical vein endothelial cells at 4 degrees C. The contribution of LAM-1 to leukocyte adhesion was only detectable when the assays were carried out under rotating (nonstatic) conditions, suggesting that LAM-1 is involved in the initial attachment of leukocytes to endothelium. In this assay at 37 degrees C, essentially all lymphocyte attachment to endothelium was mediated by LAM-1, VLA-4/VCAM-1, and the CD11/CD18 complex, whereas neutrophil attachment was mediated by LAM-1, endothelial-leukocyte adhesion molecule-1, and CD11/CD18. Thus, multiple receptors are necessary to promote optimal leukocyte adhesion to endothelium. LAM-1 also appeared to be involved in optimal neutrophil transendothelial migration using a videomicroscopic in vitro transmigration model system. LAM-1-dependent leukocyte adhesion required the induction and surface expression of a neuraminidase-sensitive molecule that was expressed for at least 24 h on activated endothelium. Expression of the LAM-1 ligand by endothelium was optimally induced by LPS and the proinflammatory cytokines TNF-alpha and IL-1 beta, whereas IFN-gamma and IL-4 induced lower levels of expression. The LAM-1 ligand on HEV and cytokine treated endothelium may be similar carbohydrate-containing molecules, because phosphomannan monoester core complex from yeast Hansenula hostii cell wall blocked binding of lymphocytes to both cell types, and identical epitopes on LAM-1-mediated lymphocyte attachment to HEV and activated endothelium. Thus, LAM-1 and its inducible endothelial ligand constitute a new pair of adhesion molecules that may regulate initial leukocyte/endothelial interactions at sites of inflammation.     

Neutrophil adhesion is impaired in the mesentery but not in the liver sinusoids of portal hypertensive rats

Altered leukocyte/cytokine response to inflammation has been observed in human and experimental portal hypertension. The aim of this study was to characterize leukocyte adhesion in portal hypertensive (PPVL) rats stimulated with endotoxin. Leukocyte rolling, adhesion, and migration assessed by intravital microscopy were impaired in mesenteric venules after lipopolysaccharide administration (150 microg/kg) in PPVL vs. sham-operated rats. Analysis of leukocyte L-selectin expression and soluble L-selectin showed that this defective adhesion was related to increased L-selectin shedding. In vitro experiments using isolated leukocytes treated with phorbol 12-myristate 13-acetate showed that monocytes and neutrophils but not lymphocytes were hyperreactive to cell activation, as measured by CD11b overexpression and increased L-selectin shedding in PPVL rats. However, neutrophil emigration in liver sinusoids and in the lung 3 h after endotoxin injection were similar in both groups of animals. Thus the alterations in leukocyte activation and adhesion molecule expression observed in this study may contribute to a better understanding of the higher susceptibility and severity of bacterial infections in cirrhotic patients with portal hypertension.     

Differential effects of CD18, CD29, and CD49 integrin subunit inhibition on neutrophil migration in pulmonary inflammation

Neutrophil migration to lung alveoli is a characteristic of lung diseases and is thought to occur primarily via capillaries rather than postcapillary venules. The role of adhesion molecules CD18 and CD29 on this migration in a mouse model of lung inflammation has been investigated. The number of neutrophils present in bronchoalveolar lavage fluid was determined 4 h after intratracheal instillation of LPS (0.1-1 microg) or murine recombinant KC (CXC chemokine, 0.03-0.3 microg). Both stimuli produced a dose-related increase in neutrophil accumulation. Intravenous anti-mouse CD18 mAb, 2E6 (0.5 mg/mouse), significantly (p < 0.001) attenuated LPS (0.3 microg)- but not KC (0.3 microg)-induced neutrophil accumulation. The anti-mouse CD29 mAb, HM beta 1-1 (0.02 mg/mouse), significantly (p < 0.05) inhibited both LPS (0.3 microg)- and KC (0.3 microg)-induced neutrophil migration. A second mAb to CD18 (GAME-46) and both F(ab')(2) and Fab of HM beta 1-1 produced similar results to those above, while coadministration of mAbs did not result in greater inhibition. Electron microscopy studies showed that CD29 was involved in the movement of neutrophils from the interstitium into alveoli. The effect of mAbs to CD49 (alpha integrin) subunits of CD29 was also examined. mAbs to CD49e and CD49f inhibited both responses, while anti-CD49b and CD49d significantly inhibited responses to KC only. These data suggest that CD29 plays a critical role in neutrophil migration in pulmonary inflammation and that CD49b and CD49d mediate CD18-independent neutrophil accumulation.     

Roles of beta 2 integrins of rat neutrophils in complement- and oxygen radical-mediated acute inflammatory injury.

The roles of beta 2 integrin molecules in neutrophil accumulation and tissue injury have been examined by the use of antibodies that are reactive with human CD11b and CD18 and cross-react with the homologous epitopes on rat neutrophils. Adherence to rat pulmonary artery endothelial cells by human neutrophils and endothelial cell killing by phorbol ester-activated human neutrophils required CD11b, CD11c, and CD18. Companion adherence studies between rat neutrophils and endothelial cells revealed a requirement for both CD11b and CD18. Neither anti-CD11b nor anti-CD18 depressed in vitro responses (O2- generation and chemotactic migration) of rat neutrophils. The accumulation of neutrophils in glycogen-induced peritoneal exudates was diminished substantially in rats treated with either anti-CD18 or anti-CD11b. In oxidant-mediated acute lung injury induced by rapid intravascular infusion of cobra venom factor, treatment of rats with either anti-CD18 or anti-CD11b significantly attenuated injury as assessed by increases in vascular permeability and hemorrhage. These protective effects correlated morphologically with diminished adhesion of neutrophils to interstitial intrapulmonary capillary endothelial cells. In studies of immune complex (BSA-anti-BSA)-induced alveolitis and dermal vasculitis, anti-CD18 had protective effects at all doses of anti-BSA employed. The protective effects of anti-CD18 correlated with diminished neutrophil accumulation in tissues at lower doses of anti-BSA. Although anti-CD11b was not effective under the same experimental conditions, intratracheal administration of this antibody conveyed protection against immune complex-induced lung injury, suggesting that both CD11b and CD18 are required for the full expression of injury. The current studies also demonstrated that when surface-bound IgG immune complexes were treated with fresh rat serum, the increment in O2- and TNF alpha generated by alveolar macrophages was suppressed by anti-CD18, but not by anti-CD11b, suggesting a heretofore unrecognized role for CD18 in the O2- and TNF-alpha responses of alveolar macrophages. Thus, neutrophil beta 2 integrins play a requisite role for the full expression of complement-dependent and oxygen radical-mediated injury of the lung and dermal vasculature.     

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion

Background

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β₂-integrin activation and function in neutrophilic firm adhesion to endothelium.

Methods

Human polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β₂-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β₂-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined.

Results

Human neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly.

Conclusions

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion.

General significance

This is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.     

Endothelial domes encapsulate adherent neutrophils and minimize increases in vascular permeability in paracellular and transcellular emigration

Local edema, a cardinal sign of inflammation associates closely with neutrophil emigration. Neutrophil emigration has been described to occur primarily through endothelial junctions (paracellular) and more rarely directly through endothelial cells (transcellular). Recently, we reported that unlike in wild-type (wt) mice, Mac-1-/- (CD11b) neutrophils predominantly emigrated transcellularly and was significantly delayed taking 20-30 min longer than the paracellular emigration (wt). In the present study we noted significant anatomical disruption of the endothelium and hypothesized that transcellular emigration would greatly increase vascular permeability. Surprisingly, despite profound disruption of the endothelial barrier as the neutrophils moved through the cells, the changes in vascular permeability during transcellular emigration (Mac-1-/-) were not increased more than in wt mice. Instead increased vascular permeability completely tracked the number of emigrated cells and as such, permeability changes were delayed in Mac-1-/- mice. However, by 60 min neutrophils from both sets of mice were emigrating in large numbers. Electron-microscopy and spinning disk multichannel fluorescence confocal microscopy revealed endothelial docking structures that progressed to dome-like structures completely covering wt and Mac-1-/- neutrophils. These domes completely enveloped the emigrating neutrophils in both wt and Mac-1-/- mice making the mode of emigration underneath these structures extraneous to barrier function. In conclusion, predominantly paracellular versus predominantly transcellular emigration does not affect vascular barrier integrity as endothelial dome-like structures retain barrier function.