ICAM-1 and CD11b/CD18 expression during acute pancreatitis induced by bile-pancreatic duct obstruction: effect of N-acetylcysteine

Different molecules are involved in the recruitment of leukocytes during inflammation. The aim was to investigate (i) the contribution of acinar cells to the overall production of ICAM-1 and (ii) the kinetics of leukocyte CD11b/CD18 expression during acute pancreatitis (AP) induced by bile-pancreatic duct obstruction (BPDO) to evaluate the contribution of both molecules to leukocyte homing. The role of reactive oxygen species (ROS) as mediators in the expression of ICAM-1 and CD11b/CD18 was examined by using N-acetylcysteine (NAC) as an antioxidant treatment. By mechanisms resistant to NAC treatment, acinar cells were able to produce ICAM-1 at first onset of AP; other cell sources contribute to maintaining increased ICAM-1 plasma levels during AP. By contrast, CD11b/CD18 was overexpressed in leukocytes in the course of AP by oxidant-dependent mechanisms. Since NAC treatment reduced neutrophil infiltration in the pancreas, we conclude that CD11b/CD18 over-expression is required for leukocyte recruitment; however, other adhesion molecules in addition to ICAM-1 seem to contribute to leukocyte homing during BPDO-induced AP.     

Monocyte chemoattractant protein-1 regulates adhesion molecule expression and cytokine production in human monocytes.

Monocytes play a critical role in defending the host against foreign organisms and in regulating the behavior of other cells. Monocytes circulate as nonadherent cells in the blood and migrate as adherent cells through tissues. Adhesion molecules mediate not only cell adhesion, but also migration, phagocytosis, and many other adhesion-dependent functions. Monocyte chemoattractant protein-1 (MCP-1) is thought to be responsible for monocyte recruitment in acute inflammatory conditions and may be an important mediator in chronic inflammation. In this study, immunofluorescence flow cytometry was used to determine whether MCP-1 can regulate the cell surface expression of adhesion molecules, particularly beta-2 and alpha-4 integrins and the leukocyte adhesion molecule-1. We found that MCP-1 induced expression of CD11c (p150,95 alpha-subunit) and CD11b (Mac-1 alpha-subunit), and caused little or no change of CD11a (lymphocyte function-associated Ag-1 alpha-subunit), very late activation Ag-4, or leukocyte adhesion molecule-1. We demonstrated that antibodies to beta-2 and alpha-4 integrins inhibited MCP-1-induced monocyte chemotaxis. We also showed that MCP-1 is capable of inducing IL-1 and IL-6, but not TNF production of monocytes. These results indicate that MCP-1 is not only a chemoattractant but also a novel cytokine with the capacity to regulate several parameters of monocyte function.     

Alteration of alveolar macrophage chemotaxis,cell adhesion,and cell adhesion molecules following ozone exposure of rats

Ozone (O₃) exposure of humans and animals induces an inflammatory response in the lung, which is associated with macrophage stimulation, release of chemotactic agents, and recruitment of polymorphonuclear leukocytes (PMNs). This study was designed to investigate the functional aspects of the macrophages that impact inflammatory processes in the lung. Macrophages recovered by bronchoalveolar lavage (BAL) from rats exposed to purified air or 0.8 ppm O₃ were studied for their chemotactic activity, adhesive interactions with alveolar epithelial cells in culture, surface morphology, and surface expression of cell adhesion molecules. The macrophages isolated from O₃-exposed rats exhibited a greater motility in response to a chemotactic stimulus than the macrophages isolated from rats exposed to purified air. The macrophages from O₃-exposed animals also displayed greater adhesion when placed in culture with epithelial cells isolated from adult rat lung (ARL-14) than the macrophages from control rats. Both chemotactic motility and cell adhesion stimulated by O₃ exposure were attenuated when the macrophages were incubated in the presence of monoclonal antibodies to leukocyte adhesion molecules, CD11b, or epithelial cell adhesion molecules, ICAM-1. Flow cytometry revealed a modest increase in the surface expression of CD11b but no change in ICAM-1 expression in macrophages from O₃-exposed rats when compared to those from the air-exposed controls. The results demonstrate an alteration of macrophage functions following O₃ exposure and suggest the dependence of these functions on the biologic characteristics, rather than the absolute expression, of the cell adhesion molecules. © 1996 Wiley-Liss, Inc.     

The anti-inflammatory effects of soluble epoxide hydrolase inhibitors are independent of leukocyte recruitment

Excess leukocyte recruitment to the lung plays a central role in the development or exacerbation of several lung inflammatory diseases including chronic obstructive pulmonary disease. Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid reported to have multiple biological functions, including blocking of leukocyte recruitment to inflamed endothelium in cell culture through reduction of adhesion molecule expression. Inhibition of the EET regulatory enzyme, soluble epoxide hydrolase (sEH) also has been reported to have anti-inflammatory effects in vivo including reduced leukocyte recruitment to the lung. We tested the hypothesis that the in vivo anti-inflammatory effects of sEH inhibitors act through the same mechanisms as the in vitro anti-inflammatory effects of EETs in a rat model of acute inflammation following exposure to tobacco smoke. Contrary to previously published data, we found that sEH inhibition did not reduce tobacco smoke-induced leukocyte recruitment to the lung. Furthermore, sEH inhibition did not reduce tobacco smoke-induced adhesion molecule expression in the lung vasculature. Similarly, concentrations of EETs greater than or equal to their reported effective dose did not reduce TNFα induced expression of the adhesion molecules. These results suggest that the anti-inflammatory effects of sEH inhibitors are independent of leukocyte recruitment and EETs do not reduce the adhesion molecules responsible for leukocyte recruitment in vitro. This demonstrates that the widely held belief that sEH inhibition prevents leukocyte recruitment via EET prevention of adhesion molecule expression is not consistently reproducible.     

Leukocyte and endothelial cell adhesion molecules in a chronic murine model of myocardial reperfusion injury

Expression of endothelial and leukocyte cell adhesion molecules is a principal determinant of polymorphonuclear neutrophil (PMN) recruitment during inflammation. It has been demonstrated that pharmacological inhibition of these molecules can attenuate PMN influx and subsequent tissue injury. We determined the temporal expression of alpha-granule membrane protein-40 (P-selectin), endothelial leukocyte adhesion molecule 1 (E-selectin), and intercellular cell adhesion molecule 1 (ICAM-1) after coronary artery occlusion and up to 3 days of reperfusion. The expression of all of these cell adhesion molecules peaked around 24 h of reperfusion. We determined the extent to which these molecules contribute to PMN infiltration by utilizing mice deficient (-/-) in P-selectin, E-selectin, ICAM-1, and CD18. Each group underwent 30 min of in vivo, regional, left anterior descending (LAD) coronary artery ischemia and 24 h of reperfusion. PMN accumulation in the ischemic-reperfused (I/R) zone was assessed using histological techniques. Deficiencies of P-selectin, E-selectin, ICAM-1, or CD18 resulted in significant (P < 0.05) attenuation of PMN infiltration into the I/R myocardium (MI/R). In addition, P-selectin, E-selectin, ICAM-1, and CD18 -/- mice exhibited significantly (P < 0.05) smaller areas of necrosis after MI/R compared with wild-type mice. These data demonstrate that MI/R induces coronary vascular expression of P-selectin, E-selectin, and ICAM-1 in mice. Furthermore, genetic deficiency of P-selectin, E-selectin, ICAM-1, or CD18 attenuates PMN sequestration and myocardial injury after in vivo MI/R. We conclude that P-selectin, E-selectin, ICAM-1, and CD18 are involved in the pathogenesis of MI/R injury in mice.     

Role of poly(ADP-ribose) synthetase in pulmonary leukocyte recruitment

During systemic inflammation, recruitment and activation of leukocytes in the pulmonary microcirculation may result in a potentially life-threatening acute lung injury. We elucidated the role of the poly(ADP-ribose) synthetase (PARS), a nucleotide-polymerizing enzyme, in the regulation of leukocyte recruitment within the lung with regard to the localization in the pulmonary microcirculation and in correlation to hemodynamics in the respective vascular segments and expression of intercellular adhesion molecule 1 during endotoxemia. Inhibition of PARS by 3-aminobenzamide reduced the endotoxin-induced leukocyte recruitment within pulmonary arterioles, capillaries, and venules in rabbits as quantified by in vivo fluorescence microscopy. Microhemodynamics and thus shear rates in all pulmonary microvascular segments remained constant. Simultaneously, inhibition of PARS with 3-aminobenzamide suppressed the endotoxin-induced adhesion molecules expression as demonstrated for intercellular adhesion molecule 1 by immunohistochemistry and Western blot analysis. We confirmed this result with the use of PARS knockout mice. The inhibitory effect of 3-aminobenzamide on leukocyte recruitment was associated with a reduction of pulmonary capillary leakage and edema formation. We first provide evidence that PARS activation mediates the leukocyte sequestration in pulmonary microvessels through upregulation of adhesion molecules. As reactive oxygen species released from leukocyte are supposed to cause an upregulation of adhesion molecules we conclude that PARS inhibition contributes to termination of this vicious cycle and inhibits the inflammatory process.     

Ninjurin1 Deficiency Attenuates Susceptibility of Experimental Autoimmune Encephalomyelitis in Mice

Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, in vivo gene deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knock-out (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis. Ninjurin1 KO mice attenuated EAE susceptibility by reducing leukocyte recruitment into the injury regions of the spinal cord and showed less adhesion of leukocytes on inflamed retinal vessels in endotoxin-induced uveitis mice. Moreover, the administration of a custom-made antibody (Ab_26–37) targeting the Ninjurin1 binding domain ameliorated the EAE symptoms, showing the contribution of its adhesion activity to leukocyte trafficking. In addition, we addressed the transendothelial migration (TEM) activity of bone marrow-derived macrophages and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO bone marrow-derived macrophages and siNinj1 Raw264.7 cells. Consistent with this, GFP-tagged mNinj1-overexpressing Raw264.7 cells increased their TEM activity. Taken together, we have clarified the contribution of Ninjurin1 to leukocyte trafficking in vivo and delineated its direct functions to TEM, emphasizing Ninjurin1 as a beneficial therapeutic target against inflammatory diseases such as multiple sclerosis.     

Human Thy-1 (CD90) on activated endothelial cells is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18)

Leukocyte recruitment in response to inflammatory signals is in part governed by interactions between endothelial cell receptors belonging to the Ig superfamily and leukocyte integrins. In our previous work, the human Ig superfamily glycoprotein Thy-1 (CD90) was identified as an activation-associated cell adhesion molecule on human dermal microvascular endothelial cells. Furthermore, the interaction of Thy-1 with a corresponding ligand on monocytes and polymorphonuclear cells was shown to be involved in the adhesion of these leukocytes to activated Thy-1-expressing endothelial cells. In this study, we have identified the specific interaction between human Thy-1 and the leukocyte integrin Mac-1 (CD11b/CD18; alphaMbeta2) both in cellular systems and in purified form. Monocytes and polymorphonuclear cells were shown to adhere to transfectants expressing human Thy-1 as well as to primary Thy-1-expressing human dermal microvascular endothelial cells. Furthermore, leukocyte adhesion to activated endothelium as well as the subsequent transendothelial migration was mediated by the interaction between Thy-1 and Mac-1. This additional pathway in leukocyte-endothelium interaction may play an important role in the regulation of leukocyte recruitment to sites of inflammation.     

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.     

Mechanisms of selective leukocyte recruitment from whole blood on cytokine-activated endothelial cells under flow conditions

Selective recruitment of eosinophils to sites of allergic and parasitic inflammation involves specific adhesion and activation signals expressed on or presented by stimulated endothelial cells. Here we examined leukocyte recruitment on cytokine-activated HUVEC under flow conditions. We perfused whole blood through a flow chamber to examine mechanisms of selective leukocyte recruitment. Although there was substantial recruitment of leukocytes on TNF-alpha-stimulated HUVEC, we found no selective accumulation of any particular leukocyte subpopulations. In contrast, fewer leukocytes were recruited to IL-4-stimulated HUVEC, but the recruitment was selective for eosinophils. We examined the role of adhesion molecules in these interactions and found that eosinophil recruitment was completely blocked with an alpha4 integrin mAb at the shear rates examined. A significant number of neutrophils were also recruited to IL-4-stimulated HUVEC, and these interactions required P-selectin and P-selectin glycoprotein ligand-1. Thus, whole blood perfusion over cytokine-activated endothelium revealed that IL-4-stimulated HUVEC support selective recruitment of eosinophils, whereas TNF-alpha-stimulated HUVEC lack selectivity for any leukocyte subclass.     

Effect of CP-96,345 on the expression of adhesion molecules in acute pancreatitis in mice

We investigated the effect of a specific neurokinin-1 receptor (NK1R) antagonist, CP-96,345, on the regulation of the expression of adhesion molecules ICAM-1, VCAM-1, E-selectin, and P-selectin as well as leukocyte recruitment during acute pancreatitis (AP). AP was induced in male Balb/C mice by 10 consecutive hourly intraperitoneal injections of caerulein. In the treatment groups, CP-96,345 was administered at 2.5 mg/kg ip either 30 min before or 1 h after the first caerulein injection. Animals were killed, and the lungs and pancreas were isolated for RNA extraction and RT-PCR or for immunohistochemical staining. mRNA expression of the four adhesion molecules was upregulated in the pancreas during AP. Treatment with CP-96,345 effectively reduced the mRNA expression of P-selectin and E-selectin but not ICAM-1 and VCAM-1. In the lung, ICAM-1, E-selectin, and P-selectin mRNA expression increased during AP. Antagonist treatment suppressed this elevation. Similar expression patterns were seen in the immunohistochemical stainings. Intravital microscopy of the pancreatic microcirculation revealed the effect of CP-96,345 on leukocyte recruitment. The present study provides important information on the relationship between NK1R activation and the regulation of adhesion molecules. Also, this study points to the differential regulation of inflammation in the pancreas and lung with AP.     

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.     

Selectin-independent leukocyte rolling and adhesion in mice deficient in E-, P-, and L-selectin and ICAM-1

To study selectin-independent leukocyte recruitment and the role of intercellular adhesion molecule-1 (ICAM-1), we generated mice lacking all three selectins and ICAM-1 (E/P/L/I-/-) by bone marrow transplantation. These mice were viable and appeared healthy under vivarium conditions, although they showed a 97% reduction in leukocyte rolling, a 63% reduction in leukocyte firm adhesion, and a 99% reduction of neutrophil recruitment in a thioglycollate-induced model of peritonitis at 4 and 24 h. Mononuclear cell recruitment was almost unaffected. All residual leukocyte rolling and most leukocyte adhesion in these mice depended on alpha(4)-integrins, but a small number of leukocytes (6% of wild-type control) still became adherent in the absence of all known rolling mechanisms (E-, P-, L-selectin and alpha(4)-integrins). A striking similarity of leukocyte adhesion efficiency in E/P/L-/- and E/P/I-/- mice suggests a pathway in which leukocyte rolling through L-selectin requires ICAM-1 for adhesion and recruitment. Comparison of our data with mice lacking individual or other combinations of adhesion molecules reveal that elimination of more adhesion molecules further reduces leukocyte recruitment but the effect is less than additive.     

Manifestations of inflammatory arthritis are critically dependent on LFA-1

Leukocyte infiltration of synovial fluid and tissues is the hallmark of inflammatory arthritis. Selectins and beta2 integrins have been implicated in the multistep process of leukocyte adhesion to vascular endothelium. However, previous work has revealed disparate requirements for leukocyte recruitments to specific anatomic locales. Moreover, the mechanisms regulating recruitment of leukocytes to the joint in inflammatory arthritis models are not fully understood. We hypothesized that beta2 integrins, expressed on leukocytes, might play a pathogenic role in synovial inflammation. Using mice deficient in all beta2 integrins (CD18 null mice), we demonstrate that expression of these heterodimeric adhesion molecules is critical for arthritis induction in the K/B x N serum transfer model. Using null-allele mice and blocking mAbs, we demonstrate specifically that CD11a/CD18 (LFA-1) is absolutely required for the development of arthritis in this model. Blocking mAbs further revealed an ongoing requirement for LFA-1 I-domain adhesive function in disease perpetuation. These findings suggest that the LFA-1 I-domain forms an attractive target for treatment of human inflammatory arthritis.     

Characterization of glycoprotein ligands for P-selectin on a human small cell lung cancer cell line NCI-H345.

P-selectin (CD62P) is a cell adhesion molecule expressed on stimulated endothelial cells and on activated platelets. It interacts with PSGL-1 (P-selectin glycoprotein ligand-1; CD162) on leukocytes and mediates recruitment of leukocytes during inflammation. P-selectin also binds to several types of cancer cells in vitro and facilitates growth and metastasis of colon carcinoma in vivo. Here we show that P-selectin, but not E-selectin, binds to NCI-H345 cells, a cell line derived from a human small cell lung cancer. EDTA or P7 (a leukocyte adhesion blocking mAb to P-selectin), but not PL5 (a leukocyte adhesion blocking mAb to PSGL-1), can inhibit this binding. P-selectin affinity chromatography can precipitate a approximately 110-kDa major band and a approximately 220-kDa minor band from [3H]-glucosamine-labeled NCI-H345 cells. No expression of PSGL-1 protein and mRNA can be detected in NCI-H345 cells. Taken together, these results suggest that NCI-H345 cells express glycoprotein ligands for P-selectin that are distinct from leukocyte PSGL-1.     

Pitavastatin prevents NMDA-induced retinal ganglion cell death by suppressing leukocyte recruitment

Excitotoxicity is a major cause of retinal ganglion cell (RGC) death during ischemic diseases such as vessel occlusion and diabetic retinopathy. However, the underlying mechanisms are not well understood. Statins, inhibitors of the HMG-CoA reductase, have neuroprotective effects in addition to their original role in lowering cholesterol. We hypothesize that pitavastatin, a recently introduced potent statin, is protective against N-methyl-d-aspartic acid (NMDA)-induced RGC death. Pitavastatin, administered by gavage, abolished NMDA-induced loss of RGCs. To elucidate the mechanisms underlying the neuroprotective effect of pitavastatin, we investigated its impact on inflammation. NMDA increased the expression of interleukin-1beta and TNF-alpha, and endothelial adhesion molecules, including ICAM-1, and induced leukocyte accumulation in the retinal vessels. Pitavastatin significantly reduced NMDA-induced leukocyte accumulation and up-regulation of endothelial adhesion molecules, whereas cytokine expression was unaffected. Systemic blockade of ICAM-1 in wild-type mice or absence of CD18 in gene-deficient (CD18(-/-)) mice significantly suppressed NMDA-induced leukocyte accumulation and RGC death. These findings suggest a novel and causative role for inflammatory leukocyte recruitment in NMDA-induced excitotoxicity. Furthermore, we show the novel neuroprotective effect of statins against excitotoxicity-induced RGC death. Statins or other anti-inflammatory agents may thus have therapeutic benefits in excitotoxicity-associated neuronal diseases through blockade of leukocyte recruitment.     

Accelerated development of arthritis in mice lacking endothelial selectins

The selectins, along with very late antigen-4 and CD44, have been implicated in mediating leukocyte rolling interactions that lead to joint recruitment and inflammation during the pathogenesis of rheumatoid arthritis. Previously, we showed that P-selectin deficiency in mice resulted in accelerated onset of joint inflammation in the murine collagen-immunized arthritis model. Here, we report that mice deficient either in E-selectin or in E-selectin and P-selectin (E/P-selectin mutant) also exhibit accelerated development of arthritis compared with wild type mice in the CIA model, suggesting that these adhesion molecules perform overlapping functions in regulating joint disease. Analyses of cytokine and chemokine expression in joint tissue from E/P-selectin mutant mice before the onset of joint swelling revealed significantly higher joint levels of macrophage inflammatory protein-1α and IL-1β compared to wild-type mice. IL-1β remained significantly increased in E/P-selectin mutant joint tissue during the early and chronic phases of arthritis. Overall, these data illustrate the novel finding that E-selectin and P-selectin expression can significantly influence cytokine and chemokine production in joint tissue, and suggest that these adhesion molecules play important regulatory roles in the development of arthritis in E/P-selectin mutant mice.     

Cytidine-5'-monophosphate-N-acetylneuraminic acid. Asialoglycoprotein sialic acid transferase activity in liver and serum of patients with juvenile hepatic cirrhosis and alpha-1-antitrypsin deficiency.

The molecular basis for the accumulation of a substance which displays the immunological reactivity of alpha-1-antitrypsin within vesicles of liver parenchymal cells of individuals with hepatic cirrhosis and serum alpha-1-antitrypsin deficiency remains unclear. We recently reported that serum from a patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis was substantially deficient in sialyltransferease (EC 2.4.99.1) an enzyme which transfers sialic acid from cytidine 5'-monophosphate-N-acetylneuraminic acid to a variety of asialoglycoprotein acceptors. In the present report we have extended these studies to include serum from five additional patients with alpha-1-antitrypsin deficiency and juvenile hepatic cirrhosis as well as a liver specimen obtained at autopsy of one of these patients. We find the sialytransferase activity in serum from six patients with alpha-1-antitrypsin deficiency and hepatic cirrhosis to be 50% of healthy pediatric control values and 30% of pediatric patients with liver disease. However, serum from family members homozygous for alpha-1-antitrypsin deficiency but without hepatic cirrhosis, and serum from patients with a variety of other kinds of liver disease, failed to exhibit the marked sialytransferase deficiency. Similar assays carried out on a homogenate of a liver sample from one patient with alpha-1-antitrypsin deficiency and hepatic cirrhosis indicated that the deficiency of sialyltransferase activity was not demonstrable in liver. Furthermore, a comparative kinetic analysis of serum and liver sialytransferase in normal and afflicted individuals failed to detect differences in substrate affinities which might account for a decrease in functional sialyltransferase capacity in individuals with alpha-1-antitrypsin deficiency and hepatic cirrhosis. These observations suggest that the serum sialyltransferase deficiency in such patients probably arises after chronic and extensive liver disease involving hepatic accumulation of alpha-1-antitrypsin rather than the enzyme deficiency being the primary cause of the hepatic cirrhosis and alpha-1-antitrypsin deficiency.     

Expression of L-selectin, but not CD44, is required for early neutrophil extravasation in antigen-induced arthritis

L (leukocyte)-selectin (CD62L) and CD44 are major adhesion receptors that support the rolling of leukocytes on endothelium, the first step of leukocyte entry into inflamed tissue. The specific contribution of L-selectin or CD44 to the regulation of cell traffic to joints in arthritis has not been investigated. We used CD44-deficient, L-selectin-deficient, and CD44/L-selectin double knockout mice to determine the requirement for these receptors for inflammatory cell recruitment during Ag-induced arthritis. Intraperitoneal immunization resulted in similar activation status and Ag-specific responses in wild-type and gene-targeted mice. However, extravasation of neutrophil granulocytes, but not the emigration of T cells, into the knee joints after intra-articular Ag injection was significantly delayed in L-selectin-deficient and double knockout mice. Intravital videomicroscopy on the synovial microcirculation revealed enhanced leukocyte rolling and diminished adherence in mice lacking either CD44 or L-selectin, but CD44 deficiency had no significant effect on the recruitment of L-selectin-null cells. Compared with wild-type leukocytes, expression of L-selectin was down-regulated in CD44-deficient cells in the spleen, peripheral blood, and inflamed joints, suggesting that reduced expression of L-selectin, rather than the lack of CD44, could be responsible for the delayed influx of granulocytes into the joints of CD44-deficient mice. In conclusion, there is a greater requirement for L-selectin than for CD44 for neutrophil extravasation during the early phase of Ag-induced arthritis.     

Macrophage migration inhibitory factor and CD74 regulate macrophage chemotactic responses via MAPK and Rho GTPase

Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment to sites of inflammation. However, whether this stems from a direct effect on leukocyte migration is unknown. Furthermore, the role of the MIF-binding protein CD74 in this response has not been investigated. Therefore, the aim of this study was to examine the contributions of MIF and CD74 to chemokine-induced macrophage recruitment. Intravital microscopy studies demonstrated that CCL2-induced leukocyte adhesion and transmigration were reduced in MIF(-/-) and CD74(-/-) mice. MIF(-/-) and CD74(-/-) macrophages also exhibited reduced chemotaxis in vitro, although CD74(-/-) macrophages showed increased chemokinesis. Reduced CCL2-induced migration was associated with attenuated MAPK phosphorylation, RhoA GTPase activity, and actin polymerization in MIF(-/-) and CD74(-/-) macrophages. Furthermore, in MIF(-/-) macrophages, MAPK phosphatase-1 was expressed at elevated levels, providing a potential mechanism for the reduction in MAPK phosphorylation in MIF-deficient cells. No increase in MAPK phosphatase-1 expression was observed in CD74(-/-) macrophages. In in vivo experiments assessing the link between MIF and CD74, combined administration of MIF and CCL2 increased leukocyte adhesion in both MIF(-/-) and CD74(-/-) mice, showing that CD74 was not required for this MIF-induced response. Additionally, although leukocyte recruitment induced by administration of MIF alone was reduced in CD74(-/-) mice, consistent with a role for CD74 in leukocyte recruitment induced by MIF, MIF-treated CD74(-/-) mice displayed residual leukocyte recruitment. These data demonstrate that MIF and CD74 play previously unappreciated roles in CCL2-induced macrophage adhesion and migration, and they indicate that MIF and CD74 mediate this effect via both common and independent mechanisms.