Mechanisms of ANCA-mediated leukocyte-endothelial cell interactions in vivo

Anti-myeloperoxidase (anti-MPO) antibodies have been implicated in the pathogenesis of small-vessel vasculitis, but the molecular mechanisms by which these antibodies contribute to disease are unknown. For determination of how anti-MPO antibodies affect inflammatory cell recruitment in small-vessel vasculitis, intravital microscopy was used to monitor leukocyte behavior in the accessible cremasteric microvessels under various experimental conditions. After local pretreatment of the cremaster muscle with cytokines (TNF-alpha, IL-1beta, or keratinocyte-derived chemokine), administration of anti-MPO IgG to wild-type mice reduced leukocyte rolling in favor of augmented adhesion to and transmigration across the endothelium. This led to a decrease in the number of systemic circulating leukocytes and, similar to the early events in the development of vasculitic lesions, an increase in leukocyte recruitment to renal and pulmonary tissue. TNF-alpha led to the greatest recruitment of inflammatory cells, and IL-1beta led to the least. When anti-CD18 was co-administered, anti-MPO IgG did not affect leukocyte rolling, adhesion, or transmigration; similarly, anti-MPO IgG did not produce these effects in Fc receptor gamma chain-/- mice. This study provides direct in vivo evidence of enhanced leukocyte-endothelial cell interactions in the presence of anti-MPO IgG and highlights the critical roles of Fcgamma receptors and beta2 integrins in mediating these interactions. In addition, it suggests that neutrophils primed by cytokines in the presence of anti-MPO IgG can have systemic effects and target specific vascular beds.     

In vivo visualization of hemodialysis-induced alterations in leukocyte-endothelial interactions

BACKGROUND: The aim of this study was to develop a model for hemodialysis (HD) in small animals using conventional dialysis equipment that would allow the intravital microscopic observation of leukocyte-endothelial interactions in vivo. METHODS: Cuprophan dialyzers were adapted to obtain a similar ratio of membrane area to blood volume as in clinical HD. A silicone ring was inserted into the dialyzer's inlet to limit the number of blood-perfused capillaries. Rabbits were dialyzed for one hour without a dialysate flow. RESULTS: Extracorporeal circulation with the cuprophan dialyzer resulted in a transient leukopenia and complement activation. At the nadir of leukopenia, leukocytes that rolled along the venular wall were scarcely observed, whereas rolling was abundant (54 +/- 9 per min) prior to extracorporeal circulation. The adhesion of leukocytes to the vascular endothelium was not induced. After 60 minutes, rolling of leukocytes was still reduced by 73 +/- 5.5%, despite the full recovery of circulating leukocyte counts. Extracorporeal circulation without a dialyzer also tended to reduce leukocyte rolling, although systemic leukocyte counts were not affected. CONCLUSIONS: The use of adapted conventional cuprophan hemodialyzers in rabbits yielded a transient leukopenia similar to that in clinical HD. Using intravital microscopy, we demonstrated impairment of leukocyte-endothelial interactions. In addition, our data indicate that tissues, in which leukocytes can roll and adhere, are not automatically sites of leukocyte sequestration during HD-induced leukopenia.     

Endothelial accumulation of hydroxyethyl starch and functional consequences on leukocyte-endothelial interactions

To date, accumulation of hydroxyethyl starch (HES) has been studied mainly in skin specimens, but there are no detailed reports available regarding starch accumulation in the endothelium. Because endothelial cells play an essential role during shock, we studied the accumulation of HES in human umbilical venous endothelial cells (HUVEC). HUVEC (n = 9) were incubated with a fluorescein-conjugated HES 200/0.5 (FITC-HES) at 0.5-20 mg/ml for 1-72 h. FITC-HES was internalized dose- and time-dependently by pinocytosis into secondary lysosomes. Asymptotic elimination curves showed that 50% of the formerly ingested molecules could not be eliminated. Despite accumulation, starch molecules did not attenuate the expression of E-selectin, ICAM-1 or VCAM-1 on TNF-alpha-activated HUVEC. However, apart from adhesion molecule expression, perfusion studies showed that HES reduced neutrophil adhesion by direct inhibition of integrin-mediated interactions.     

Role of inflammation (leukocyte-endothelial cell interactions) in vasospasm after subarachnoid hemorrhage

BackgroundDelayed vasospasm is the leading cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (aSAH). This phenomenon was first described more than 50 years ago, but only recently has the role of inflammation in this condition become better understood.MethodsThe literature was reviewed for studies on delayed vasospasm and inflammation.ResultsThere is increasing evidence that inflammation and, more specifically, leukocyte-endothelial cell interactions play a critical role in the pathogenesis of vasospasm after aSAH, as well as in other conditions including meningitis and traumatic brain injury. Although earlier clinical observations and indirect experimental evidence suggested an association between inflammation and chronic vasospasm, recently direct molecular evidence demonstrates the central role of leukocyte-endothelial cell interactions in the development of chronic vasospasm. This evidence shows in both clinical and experimental studies that cell adhesion molecules (CAMs) are up-regulated in the perivasospasm period. Moreover, the use of monoclonal antibodies against these CAMs, as well as drugs that decrease the expression of CAMs, decreases vasospasm in experimental studies. It also appears that certain individuals are genetically predisposed to a severe inflammatory response after aSAH based on their haptoglobin genotype, which in turn predisposes them to develop clinically symptomatic vasospasm.ConclusionBased on this evidence, leukocyte-endothelial cell interactions appear to be the root cause of chronic vasospasm. This hypothesis predicts many surprising features of vasospasm and explains apparently unrelated phenomena observed in aSAH patients. Therapies aimed at preventing inflammation may prevent and/or reverse arterial narrowing in patients with aSAH and result in improved outcomes.     

Phosphate may promote CKD progression and attenuate renoprotective effect of ACE inhibition

Phosphate may promote the onset and progression of chronic nephropathies. Here we evaluated the relationships between baseline serum phosphate levels, disease progression, and response to ACE inhibition in 331 patients with proteinuric nephropathies in the prospective Ramipril Efficacy In Nephropathy (REIN) trial. Independent of treatment, patients with phosphate levels in the highest two quartiles progressed significantly faster either to ESRD or to a composite endpoint of doubling of serum creatinine or ESRD compared with patients with phosphate levels below the median (P < 0.001). Results were similar when we analyzed phosphate as a continuous variable (P ≤ 0.004). The renoprotective effect of ramipril decreased as serum phosphate increased (P ≤ 0.008 for interaction); this modification of the treatment effect by phosphate persisted despite adjusting for potential confounders such as GFR and urinary protein. In summary, these data suggest that phosphate is an independent risk factor for progression of renal disease among patients with proteinuric CKD, and high levels of phosphate may even attenuate the renoprotective effect of ACE inhibitors. Future trials should test whether reducing serum phosphate improves renal outcomes and optimizes the renoprotective effect of ACE inhibition.     

Hypertonic saline resuscitation attenuates neutrophil lung sequestration and transmigration by diminishing leukocyte-endothelial interactions in a two-hit model of hemorrhagic shock and infection

BACKGROUND: Hypertonic saline (HTS) attenuates polymorphonuclear neutrophil (PMN)-mediated tissue injury after hemorrhagic shock. We hypothesized that HTS resuscitation reduces early in vivo endothelial cell (EC)-PMN interactions and late lung PMN sequestration in a two-hit model of hemorrhagic shock followed by mimicked infection. METHODS: Thirty-two mice were hemorrhaged (40 mm Hg) for 60 minutes and then given intratracheal lipopolysaccharide (10 microg) 1 hour after resuscitation with shed blood and either HTS (4 mL/kg 7.5% NaCl) or Ringer's lactate (RL) (twice shed blood volume). Eleven controls were not manipulated. Cremaster intravital microscopy quantified 5-hour EC-PMN adherence, myeloperoxidase assay assessed lung PMN content (2 1/2 and 24 hours), and lung histology determined 24-hour PMN transmigration. RESULTS: Compared with RL, HTS animals displayed 55% less 5-hour EC-PMN adherence (p = 0.01), 61% lower 24-hour lung myeloperoxidase ( p= 0.007), and 57% lower mean 24-hour lung histologic score ( p= 0.027). CONCLUSION: Compared with RL, HTS resuscitation attenuates early EC-PMN adhesion and late lung PMN accumulation in hemorrhagic shock followed by inflammation. HTS resuscitation may attenuate PMN-mediated organ damage.     

Reduction by superoxide dismutase of leukocyte-endothelial adherence after liver transplantation.

One hour after orthotopic rat liver transplantation, hepatic microcirculation and adhesion characteristics of leukocytes to the endothelial wall were studied with intravital microscopy. Cold storage for 1 and 8 hours in Euro-Collins solution resulted in reduction of perfused sinusoids to 83% and 48% and a decrease of leukocyte velocity from 417 microns/sec in controls to 311 microns/sec and 269 microns/sec, respectively. Additionally, the number of permanently adherent leukocytes rose from 4% in controls to 33% and 43% after cold storage for 1 and 8 hours. Intravenous injection of the free radical scavenger human recombinant superoxide dismutase (40 mg/kg) during reperfusion resulted in marked improvement of the hepatic microcirculation after both 1 and 8 hours of cold storage (91% and 69% perfused sinusoids; 420 microns/sec and 350 microns/sec leukocyte velocity; p less than 0.05). Furthermore, the percentage of permanently adherent leukocytes decreased to 13% and 28% after 1 and 8 hours of cold ischemia, respectively. These results support the hypothesis that oxygen-derived free radicals contribute to leukocyte adherence and microcirculatory failure after cold liver ischemia and transplantation. Thus, application of oxygen free radical scavengers during liver transplantation procedures might be useful to improve graft function.     

Nonsteroidal anti-inflammatory drugs attenuate epidermal growth factor-induced proliferation independent of prostaglandin synthesis inhibition.

BACKGROUND: The mechanism(s) whereby nonsteroidal anti-inflammatory drugs (NSAIDs) attenuate colorectal tumor growth remains poorly understood. This study determined if NSAIDs decreased epidermal growth factor (EGF)-induced proliferation in human colonic tumor (Caco-2) cells and whether this process involved the inhibition of prostaglandin (PG) synthesis. METHODS: Caco-2 cells were serum-starved (48 h) and subsequently treated (48 h) with either serum-free media or EGF (10 ng/ml) +/- physiologic and noninjurious (as determined by LDH release) concentrations of aspirin, indomethacin, and ibuprofen. PG synthesis was measured by EIA. Proliferation was quantitated with two assays: cellular protein and nucleic acid content. RESULTS: NSAID treatment did not inhibit growth in cells treated with only serum-free media. Cells exposed to EGF demonstrated a significant increase in PGE2, protein, and nucleic acid. Levels of other eicosanoids (PGI2, TXA2) were minimal both before and after EGF treatment. Despite varying degrees of PGE2 inhibition, each NSAID group equally attenuated EGF-induced protein and nucleic acid synthesis. The correlation between PGE2 levels and protein (R2 = 0.56) or nucleic acid (R2 = 0.54) was poor. Finally, the addition of a physiologically appropriate concentration of exogenous PGE2 failed to reverse NSAID-induced growth inhibition. CONCLUSION: These data suggest that NSAIDs, independent of PG synthesis inhibition, attenuate EGF-induced proliferation in Caco-2 cells. This may provide one explanation for how NSAIDs limit colonic neoplasia.     

Propacetamol augments inhibition of platelet function by diclofenac in volunteers

Background. Acetaminophen (paracetamol) enhances the analgesiceffect of non-steroidal anti-inflammatory drugs (NSAIDs). Acetaminophenis a weak inhibitor of cyclooxygenase (COX), and its combinationwith an NSAID may augment COX inhibition-related side effects. Methods. Ten healthy male volunteers (21–30 yr) were givendiclofenac 1.1 mg kg^–1 alone, a combination of propacetamol30 mg kg^–1 (which is hydrolysed to 50% acetaminophen)and diclofenac 1.1 mg kg^–1 or placebo intravenously ina double blind, crossover study. Platelet function was assessedat 5 min, 90 min and 22–24 h by photometric aggregometry,platelet function analyser (PFA-100^TM) and by measuring therelease of thromboxane B₂ (TxB₂). Analgesia was assessed withthe cold pressor test. Results. Platelet aggregation induced with arachidonic acidwas fully inhibited by both diclofenac alone and the combinationat the end of the 30-min drug infusion. Propacetamol augmentedthe inhibition by diclofenac at 90 min (P=0.014). At 22–24h, platelet function had fully recovered. TxB₂ release was inhibitedby the combination of propacetamol and diclofenac at 90 minin comparison with diclofenac alone (P=0.027). PFA-100^TM detectedno difference in platelet function between these two groups.No analgesic effect was detected with the cold pressor test. Conclusions. The combination of propacetamol and diclofenacinhibits platelet function more than diclofenac alone. Thisshould be considered when assessing the risk of surgical bleeding. Br J Anaesth 2003; 91: 357–62     

Blood volume expansion with hyperoncotic colloids deteriorates allograft function in a canine model of renal transplantation

PURPOSE: We investigated the effects of acute maximal hydratation with hemoce (H) and dextran-40 (D40) on the postoperative graft function, following renal transplantation (RT) in a canine model. METHODS: After induction of anesthesia with pentobarbital (5 mg/kg), 18 beagle dogs were randomized to receive either saline solution to increase the central venous pressure (CVP) to 5 mm Hg (GI); H solution to increase the CVP to 10 mm Hg (GII); or D40 to achieve 15 mm Hg (GIII), before reperfusion. A pulmonary artery catheter was used to measure CVP, mean pulmonary artery pressure, and cardiac output (CO). The surgical procedure consisted of autotransplantation of the dog's left kidney an hour prior to cold ischemia with University of Wisconsin solution, followed by contralateral nephrectomy. Diuresis, creatinine (Cr), and BUN levels were measure at 24 hours before RT, as well as 24, 48, and 72 hours after the procedure. RESULTS: Only in the treated groups did cardiac filling pressures and CO increase as a result of hydration. Only in the GI group did serum Cr and blood urea nitrogen significantly peak at the second postoperative day while it continued to increase at two (GII) and three (GIII) times greater than GI on the third day. Histological examination showed osmotic nephrosis like-lesions only among treated grafts. CONCLUSION: We concluded that maximal hydration with H and D40 colloid deteriorated postoperative graft function after RT. We believe that in the future the effects of any colloid solution should be tested in an animal model in the fashion as we have described, in order to know which one, and at what dose, is the safest to improve kidney allograft outcome.     

Cardiopulmonary bypass reduces peripheral microvascular contractile function by inhibition of mitogen-activated protein kinase activity

BACKGROUND: Mitogen-activated protein kinases (MAPK) have been implicated in pathophysiologic responses to cardiopulmonary bypass (CPB). MAPK are deactivated by phosphatases, such as MAPK phosphatase-1 (MKP-1). We hypothesized that MAPK mediate peripheral microvascular contractile dysfunction caused by CPB in humans. METHODS: Skeletal muscle was harvested before and after CPB. Protein levels of MKP-1 and activated extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 were measured. MKP-1 gene expression was measured. Peripheral microvessel responses to vasopressors were studied by videomicroscopy. Contractile function also was measured after MAPK inhibition with PD98059 (ERK1/2) and SB203580 (p38). ERK1/2, p38, and MKP-1 were localized by immunohistochemistry and in situ hybridization. RESULTS: ERK1/2 and p38 activity was decreased in peripheral tissue after CPB. MKP-1 was increased after CPB. Contractile responses of peripheral arterioles to phenylephrine and vasopressin were decreased after CPB. Microvessel reactivity also was reduced after treatment with PD98059 and SB203580. ERK1/2, p38, and MKP-1 localized to peripheral arterioles in tissue sections. CONCLUSIONS: CPB reduces ERK1/2 and p38 activity in peripheral tissue, potentially by MKP-1. Contractile responses of peripheral arterioles to phenylephrine and vasopressin are dependent on ERK1/2 and p38 and are decreased after CPB. These results suggest that alterations in MAPK pathways in part regulate peripheral microvascular dysfunction after CPB in humans.     

Characterization of inhibition of platelet function by paracetamol and its interaction with diclofenac in vitro

BACKGROUND: Paracetamol (acetaminophen) is an effective analgesic and a weak inhibitor of cyclo-oxygenase (COX). Clinically paracetamol is often used together with traditional NSAIDs, which are strong inhibitors of COX. We studied binding of paracetamol to COX and its action on platelet function together with diclofenac. METHODS: Blood was collected from healthy donors and platelet function was assessed by photometric aggregometry, a platelet function analyser (PFA-100, Dade Behring, Deerfield, IL) and by measuring the release of thromboxane B(2) (TxB(2)), the stable metabolite of thromboxane A(2), after addition of paracetamol (10-80 microg ml(-1)). A concentration-inhibition relationship was established and the inhibition coefficient (K(i)) demonstrating 50% binding to COX was determined using a Schild-plot. Interaction of paracetamol (5-20 microg ml(-1)) and diclofenac (0.1-0.8 microg ml(-1)) was determined and an isobolographic analysis was performed. RESULTS: Paracetamol added to platelet-rich plasma (PRP) caused a concentration-dependent inhibition of platelet function. Photometric aggregometry and TxB(2) release was significantly inhibited by paracetamol from 10 microg ml(-1) onwards. The PFA-100 closure time was significantly prolonged by paracetamol at a high concentration only. K(i) was 15.2 microg ml(-1) with a 95% confidence interval of 11.8-18.6 microg ml(-1). Inhibition of aggregation by diclofenac was augmented by paracetamol. Isobolographic analysis showed synergism. CONCLUSIONS: The 95% confidence interval of K(i) equals the antipyretic plasma concentration of paracetamol, i.e. 10-20 microg ml(-1). High doses of paracetamol and a combination of diclofenac and paracetamol cause platelet inhibition and thus may increase risk of surgical bleeding.     

老年肺部感染患者细胞免疫功能及整合素表达研究

目的 研究老年肺部感染患者细胞免疫功能及整合素表达变化情况,为临床诊疗提供依据.方法 采用流式细胞术检测老年肺部感染患者及健康老年人的细胞免疫表型(CD3、CD4、CD8、CD4/CD8、CD16和CD19)以及整合素β1(CD29)、β2(CD18)和β3(CD61)的表达情况.结果老年肺部感染患者CD3、CD4、CD4/CD8与CD19表达水平均较老年健康组显著下降(P< 0.01);整合素β1和β3表达水平显著升高(P< 0.01).结论 老年肺部感染的发生、发展、转归与机体细胞免疫功能紊乱及整合素的高表达有关,通过这些指标的监测可以为临床诊断、治疗和预防提供有效的参考.     

Verapamil inhibition of lymphocyte proliferation and function in vitro

Calcium is a divalent cation which acts both as a cofactor for critical protein function and as a "second signal" to trigger cytosol events after membrane depolarization. The presence of calcium is critical to T-lymphocyte function. Verapamil is a calcium channel blocker at the membrane level and may affect cytosol calcium-calmodulin as well. Verapamil effect was measured on two assays of lymphocyte proliferation (concanavalin A and phytohemagglutinin lectin and alloantigen stimulated) and on cell-mediated cytotoxicity. Verapamil inhibited proliferation at 10(-5) M doses and abrogated it at 10(-4) M. Verapamil interferes with critical calcium-mediated events in T lymphocyte proliferation and function. T-lymphocyte calcium channel blockade may be an effective immunosuppressive strategy.     

Evidence for nongastrin-mediated somatostatin inhibition of parietal cell function

Somatostatin (SRIF), a tetradecapeptide, has been reported to suppress gastrin release and hence inhibit acid secretion in vivo. This study was performed to determine whether SRIF has any direct effect on parietal cell (PC). Isolated gastric cells were prepared by collagenase digestion and calcium chelation of rabbit fundic mucosa. PC enrichment (75% +/- 5%) was accomplished by velocity sedimentation with an elutriator rotor. Acid, as assessed by the accumulation of 14C-aminopyrine (AP) and macromolecular (intrinsic factor [IF]) secretion were used as markers of PC function. Cells were stimulated with histamine (H) (10(-6) mol/L). SRIF (10(-10) to 10(-6) mol/L) significantly inhibited H-stimulated 14C-AP accumulation (p less than 0.05). Inhibition of H-stimulated IF release was less sensitive, occurring at 10(-8) and 10(-7) mol/L (p less than 0.05), and loss of inhibition was observed at 10(-6) mol/L (p less than 0.05). These results demonstrate a direct inhibitory action of SRIF on PC secretion. The difference in inhibitory effect on IF and proton secretion is consistent with the postulation that SRIF may function at more than one site within the PC.