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Recent advances using transgenic animals or exogenous complement inhibitors have demonstrated prevention of hyperacute rejection of vascularized organs, but not graft loss due to acute vascular rejection. Using various wild-type and cytokine-deficient mice strains, we have examined the mechanisms of acute vascular rejection. C57BL/6 mice deficient in interleukin12 or gamma interferon showed faster acute vascular rejection than did wild-type mice. Furthermore, mice defective in B-cell development showed no acute vascular rejection. These results demonstrate that the axis of interleukin 12 and gamma interferon provides a survival advantage in vascularized xenografts by delaying or preventing acute vascular rejection caused by a B cell-dependent mechanism.

In the present study, we explored the molecular mechanisms by which bacterial endotoxin (LPS) mediates the down-regulation of CCR2 receptors on human monocytes. We found that LPS induced a marked reduction in CCR2 cell surface protein levels which was blocked by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A. The effector mechanism underlying LPS-induced CCR2 down-modulation appears to involve the enzymatic activity of proteinases since Western blot analysis of LPS-stimulated monocytes revealed the degradation of a 38-kDa species corresponding to the CCR2B monomer. In RBL cells expressing the CCR2B-green fluorescent protein (GFP) fusion chemokine receptor, LPS stimulated the internalization and degradation of CCR2. The serine proteinase inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone blocked LPS-induced down-modulation of CCR2 in monocytes and CCR2B-GFP in RBL cells. This work describes a previously uncharacterized mechanism for CC chemokine receptor down-modulation that is dependent upon tyrosine kinase activation and serine proteinase-mediated receptor degradation and may provide further insight into the mechanisms of leukocyte regulation during immunological and inflammatory responses.

All leukocytes express the cell surface glycoprotein CD45, which has intrinsic intracellular protein tyrosine phosphatase activity. CD45 is known to play a regulatory role in activation-induced signaling in lymphocytes; however, little is known of its role in non-lymphoid leukocytes. Therefore, we examined the potential effect of CD45 on chemokine-induced signaling in human neutrophils (polymorphonuclear cells, PMN). Treating isolated PMN for 2 h with an anti-CD45RB antibody (Bra11) down-modulated expression of the chemokine receptors CXCR1 and CXCR2 to 44 +/- 10% and 47 +/- 9% of their respective controls. The tyrosine kinase inhibitors genistein and herbimycin A significantly inhibited the Bra11-induced down-modulation of CXCR1 and CXCR2. Furthermore, Bra11-treated PMN were functionally inhibited in their capacity to exhibit IL-8-induced transient intracellular Ca2+ increases. Selected targeting of CXC receptors is indicated by the fact that N-formyl-Met-Leu-Phe (fMLP) receptor expression and function were not lost following Bra11 treatment. The effect of Bra11 on IL-8-mediated function and receptor expression was paralleled by decreased tyrosine phosphorylation of a 54- to 60-kDa protein. These findings indicate that CD45 can act to modulate PMN responses to chemokines; thus agents regulating CD45 can potentially modulate leukocyte traffic and may represent a novel therapeutic approach towards the treatment of inflammatory diseases.

The neutrophil-specific G-protein–coupled chemokine receptors, CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are not well defined, although previous studies have suggested a process of ligand-promoted internalization as a putative regulatory pathway. Herein, we provide evidence for two distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy data showed a redistribution of CXCR1 expression from the cell surface of neutrophils to internal compartments after stimulation with IL-8, whereas stimulation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor- (TNF-) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investigate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-–induced downmodulation of IL-8 receptors, we tested a panel of proteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF- was most dramatically inhibited by metalloproteinase inhibitors; 1,10-phenanthroline and EDTA significantly attenuated LPS- and TNF-–induced loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibitors also blocked the release of CXCR1 cleavage fragments into the cell supernatants of LPS- and TNF-–stimulated neutrophils. In addition, while treatment of neutrophils with LPS and TNF- inhibited IL-8 receptor–mediated calcium mobilization and IL-8–directed neutrophil chemotaxis, both 1,10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8–mediated downmodulation of CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, these findings may provide further insight into the mechanisms of leukocyte regulation during immunologic and inflammatory responses.

The neutrophil-specific G-protein–coupled chemokine receptors, CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are not well defined, although previous studies have suggested a process of ligand-promoted internalization as a putative regulatory pathway. Herein, we provide evidence for two distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy data showed a redistribution of CXCR1 expression from the cell surface of neutrophils to internal compartments after stimulation with IL-8, whereas stimulation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor- (TNF-) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investigate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-–induced downmodulation of IL-8 receptors, we tested a panel of proteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF- was most dramatically inhibited by metalloproteinase inhibitors; 1,10-phenanthroline and EDTA significantly attenuated LPS- and TNF-–induced loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibitors also blocked the release of CXCR1 cleavage fragments into the cell supernatants of LPS- and TNF-–stimulated neutrophils. In addition, while treatment of neutrophils with LPS and TNF- inhibited IL-8 receptor–mediated calcium mobilization and IL-8–directed neutrophil chemotaxis, both 1,10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8–mediated downmodulation of CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, these findings may provide further insight into the mechanisms of leukocyte regulation during immunologic and inflammatory responses.

Mechanisms of induction of renal allograft tolerance in CD45RB-treated mice.BackgroundRejection is the most significant problem in the field of transplantation. The current goal of transplant immunology is to develop better immunotherapeutic protocols that are aimed at specifically suppressing alloreactivity and preserving an otherwise intact immune system. We have previously shown that mice will accept renal allografts indefinitely with normal renal function after two injections of a monoclonal antibody to the CD45RB protein. Furthermore, this antibody will reverse acute rejection when therapy is delayed until day 4 and will still induce tolerance. The mechanisms of this therapeutic benefit are not known.MethodsBALB/C mice were used as recipients of major multiple histocompatibility complex-mismatched kidneys using C57BL/6 as donors. Immunoperoxidase microscopy and Northern blots for cytokine gene expression were used to study the renal allografts. Fluorescence-activated cell sorter (FACS) analyses of peripheral blood lymphocytes were performed. Phosphotyrosine peptide phosphatase assays were performed on splenic lymphocyte membranes.ResultsA CD45RB monoclonal antibody (MB23G2) induced tolerance and partially depletes peripheral blood lymphocytes. A therapeutically ineffective CD45RB monoclonal antibody (MB4B4) merely coated the circulating lymphocytes. Furthermore, MB23G2 stimulated more tyrosine phosphatase activity than MB4B4 in mouse T-cell membranes.ConclusionsThe clearance of peripheral blood lymphocyte populations and stimulation of protein tyrosine phosphatase activity may be important in the mechanism of tolerance induction by CD45RB therapy, which may be clinically relevant in the therapy of organ rejection in humans.

Staphylococcal superantigens (SAgs) are very potent T cell mitogens, but they can also activate monocytes by binding directly to MHC class II molecules in a manner independent of TCR coengagement. Induction of proinflammatory cytokines and chemokine expression in monocytes by superantigens has recently been reported. Here we report that superantigen stimulation of human peripheral blood monocytes results in a rapid, dose-dependent, and specific down-regulation of chemokine (macrophage inflammatory protein-1α (MIP-1α), monocyte chemotactic protein-1 and MIP-1β) binding sites (e.g., CCR1, CCR2, and CCR5), which correlates with a concomitant hyporesponsiveness of human monocytes to these CC chemokine ligands. This down-regulation occurs 15–30 min following superantigen stimulation and is specific to chemokine receptors, in that binding and responsiveness of monocytes to the chemoattractant formyl-tripeptide FMLP are not affected. We further demonstrate that SAg-induced down-modulation of chemokine binding and monocyte hyporesponsiveness to the chemokines MIP-1α, monocyte chemotactic protein-1, and MIP-1β is mediated through cellular protein tyrosine kinases, and the down-modulation can be mimicked by an MHC class II-specific mAb. Additionally, our observations indicate that SAg-induced loss of chemokine binding and monocyte responsiveness is probably mediated by secreted serine proteinases. Bacterial SAg-induced down-modulation of chemokine responsiveness represents a previously unrecognized strategy by some bacteria to subvert immune responses by affecting the intricate balance between chemokine and chemokine receptor expression and function.

The expression of the seven-transmembrane domain chemokine receptors CXCR1 and CXCR2 modulates neutrophil responsiveness to the chemoattractant IL-8 and a number of closely related CXC chemokines. In the present study, we investigated the mechanism by which bacterial LPS induces the down-modulation of IL-8 responsiveness and CXCR1 and CXCR2 expression on human neutrophils. Treating neutrophils with LPS reduced IL-8R expression to 55 ± 5% of the control within 30 min and to 23 ± 2% within 1 h of stimulation. Furthermore, this down-modulation could not be attributed to increased concentrations of IL-8, TNF-α, or IL-1β, since ELISA studies indicated that LPS-stimulated neutrophils did not release detectable amounts of these proteins before 2 h poststimulation. The tyrosine kinase (TK) inhibitors genistein and herbimycin A attenuated the LPS-mediated down-modulation of CXCR1 and CXCR2, indicating that the activation of a TK is required for LPS to mediate its effect. The effect of LPS on receptor expression paralleled the hyperphosphorylation of the protein TK p72syk. Although IL-8 induced a comparable down-modulation of CXCR1 and CXCR2, TK inhibitors did not attenuate this effect. These studies provide the first evidence of an agonist-independent, TK-dependent pathway of chemokine receptor regulation by endotoxin.

To determine whether surgical pericardiectomy is a safe and effective alternative to medical management for chronic relapsing pericarditis.Retrospective review of 184 patients presenting to the Mayo Clinic in Rochester, Minnesota, from January 1, 1994, through December 31, 2005, with persistent relapsing pericarditis identified 58 patients who had a pericardiectomy after failed medical management and 126 patients who continued with medical treatment only. The primary outcome variables were in-hospital postoperative mortality or major morbidity, all-cause death, time to relapse, and medication use.Mean ± SD follow-up was 5.5 ± 3.5 years in the surgical group and 5.4 ± 4.4 years in the medical treatment group. At baseline, patients in the surgical group had higher mean relapses (6.9 vs 5.5; P=.01), were more likely to be taking colchicine (43.1% [n=25] vs 18.3% [n=23]; P=.002) and corticosteroids (70.7% [n=41] vs 42.1% [n=53]; P.001), and were more likely to have undergone a prior pericardiotomy (27.6% [n=16] vs 11.1% [n=14]; P=.003) than the medical treatment group. Perioperative mortality (0%) and major morbidity (3%; n=2) were minimal. Kaplan-Meier analysis revealed no differences in all-cause death at follow-up (P=.26); however, the surgical group had a markedly decreased relapse rate compared with the medical treatment group (P=.009). Medication use was notably reduced after pericardiectomy.In patients with chronic relapsing pericarditis in whom medical management has failed, surgical pericardiectomy is a safe and effective method of relieving symptoms.

We sought to propose a magnetic resonance (MR) imaging-derived index of biventricular interdependence as a diagnostic parameter to distinguish patients with surgically-confirmed pericardial constriction from those without. Free-breathing real time MR pulse sequences of seventeen subjects with surgically proven constrictive pericarditis and thirty-five patients referred for clinically-indicated cardiac MR examinations but without documented constriction were analyzed using a novel index of biventricular interdependence. Cross-sectional biventricular areas at end diastole using the epicardial surface were traced at the mid left ventricular level at end-inspiration and end-expiration and an index of biventricular interdependence, defined as the ratio of (biventricular end-diastolic area at end-inspiration)/(biventricular end-diastolic area at end-expiration) was calculated for each subject. The mean index for both groups was calculated and results were statistically compared. The index of biventricular interdependence approximated unity (mean index 1.03 ± 0.03 SD) in patients with surgically confirmed pericardial constriction, indicating similar biventricular area at end-inspiration and end-expiration, and was significantly lower than in individuals without constrictive pericarditis (mean index 1.28 ± 0.10 SD; p < 0.0001). The MR-derived index of biventricular interdependence was significantly different between subjects with surgically-confirmed pericardial constriction and subjects where pericardial constraint was not suspected and may serve as a useful metric in the hemodynamic assessment of patients with a potential diagnosis of constrictive pericarditis.

Pericardial diseases can present clinically as acute pericarditis, pericardial effusion, cardiac tamponade, and constrictive pericarditis. Patients can subsequently develop chronic or recurrent pericarditis. Structural abnormalities including congenitally absent pericardium and pericardial cysts are usually asymptomatic and are uncommon. Clinicians are often faced with several diagnostic and management questions relating to the various pericardial syndromes: What are the diagnostic criteria for the vast array of pericardial diseases? Which diagnostic tools should be used? Who requires hospitalization and who can be treated as an outpatient? Which medical management strategies have the best evidence base? When should corticosteroids be used? When should surgical pericardiectomy be considered? To identify relevant literature, we searched PubMed and MEDLINE using the keywords diagnosis, treatment, management, acute pericarditis, relapsing or recurrent pericarditis, pericardial effusion, cardiac tamponade, constrictive pericarditis, and restrictive cardiomyopathy. Studies were selected on the basis of clinical relevance and the impact on clinical practice. This review represents the currently available evidence and the experiences from the pericardial clinic at our institution to help guide the clinician in answering difficult diagnostic and management questions on pericardial diseases.

The American College of Cardiology Foundation/American Society of Nuclear Cardiology appropriateness criteria document assigns single photon emission computed tomography myocardial perfusion imaging (SPECT MPI) a rating of uncertain for detection and risk assessment of coronary artery disease (CAD) in asymptomatic patients at moderate risk. The nuclear cardiology database was used to identify 260 asymptomatic patients (67 ± 8 years, 72% men) without known CAD who were at moderate CAD risk according to the Framingham risk score. SPECT MPI images were categorized using the summed stress score (SSS). Mean follow-up 9.9 ± 3.0 years. Abnormal SPECT MPI scans were present in 142 patients (55%). By SSS categories, SPECT scans were low-risk in 67%, intermediate-risk in 20%, and high-risk in 13% of patients. Overall survival at 10 years was 79%, significantly better than the age- and gender-matched Minnesota general population (P

Background. Embolism is a dreaded complication of infective endocarditis (IE). Currently, antimicrobial therapy is the only medical intervention proven to decrease the risk of embolism associated with IE. We hypothesized that, because platelet aggregation is operative in the pathogenesis of vegetation formation, embolism associated with IE should occur less frequently among patients who have received prior, continuous daily antiplatelet therapy for noninfectious reasons. Methods. We studied a retrospective cohort of adult patients with a diagnosis of IE who presented to the Mayo Clinic (Rochester, MN) during 1980-1998. The cohort was divided into 2 groups on the basis of whether they had received continuous daily antiplatelet therapy for at least 6 months prior to the time of hospitalization for IE. Antiplatelet therapy included aspirin, dipyridamole, clopidogrel, ticlopidine, or any of combination of these agents. The primary end point was a symptomatic embolic event that occurred prior to or during hospitalization. Multivariable logistic regression was used to assess the impact of continuous daily antiplatelet therapy on risk of symptomatic emboli associated with IE. Results. One hundred forty-seven (24.5%) of 600 patients experienced a symptomatic embolic event; the most common embolic manifestation was stroke (in 48.2% of patients). Embolic events occurred significantly less often among those who had received prior, continuous daily antiplatelet therapy (12.0% of patients who had received therapy vs. 27.8% patients who had not receive therapy; P

The functional role of neutrophils during acute inflammatory responses is regulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) that are rapidly desensitized and internalized upon binding their cognate chemokine ligands. The efficient re-expression of CXCR1 on the surface of neutrophils following agonist-induced internalization suggests that CXCR1 surface receptor turnover may involve regulatory pathways and intracellular factors similar to those regulating beta2-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligand-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two different cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimulation promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalization in HEK 293 cells required co-expression of G protein-coupled receptor kinase 2 and beta-arrestin proteins. The importance of beta-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative beta-arrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalization in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Agonist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin-coated vesicles. Our data provides a direct link between agonist-induced internalization of CXCR1 and a requirement for G protein-coupled receptor kinase 2, beta-arrestins, and dynamin during this process.