Apoptosis and chemokine induction after renal ischemia-reperfusion.

BACKGROUND: One of the earliest prerequisites for the development of inflammation after ischemia-reperfusion (I/R) is local chemokine expression. We recently demonstrated that apoptosis, characterized by intracellular caspase-activation, contributes to the development of inflammation after I/R. METHODS: The contribution of apoptosis was investigated using the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F in a murine model of renal I/R. Renal expression of the chemokines macrophage inflammatory protein-2 (MIP-2) and KC was studied using RT-PCR and immunohistology. Measuring myeloperoxidase activity and serum ureum and creatinine levels assessed neutrophil influx and kidney dysfunction. RESULTS: We demonstrate renal up-regulation of KC and MIP-2 after 1 to 16 hr of reperfusion. Treatment with the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F effectively prevented I/R-induced renal apoptosis, KC, and MIP-2 up-regulation after 2 hr of reperfusion as well as neutrophil influx and functional impairment after 24 hr of reperfusion. CONCLUSIONS: These data for the first time show that chemokine induction following I/R is dependent on caspase activation.

Activated caspase-1 is not a central mediator of inflammation in the course of ischemia-reperfusion.

BACKGROUND: Upon transplantation, donor organs subjected to prolonged ischemia suffer from reperfusion injury. Recent observations suggest that caspase activation is involved in inducing the deleterious inflammatory reaction that mediates reperfusion injury. Release of cytokines like interleukin (IL)-1 and IL-18 may occur during apoptosis through activation of caspase-1/IL-1beta-converting enzyme. We hypothesized that caspase-1 activation is a key event in apoptosis/ caspase-dependent inflammation during the development of renal reperfusion injury. METHODS: Caspase-1-/-, caspase-1+/+ as well as Swiss mice were subjected to 45 min of renal ischemia and 24 hr of reperfusion. Animals were administered agents capable of neutralizing the pro-inflammatory activation products of caspase-1 (IL-1 receptor antagonist, anti-IL-1 receptor antibody, and anti-IL-18 antibody). The extent of renal functional deterioration, inflammation, and apoptosis were compared. RESULTS: No improvement in renal function as reflected by serum ureum and creatinine were found in caspase-1-/- mice as compared to wild type controls. Caspase-1-/- mice showed slightly attenuated renal inflammation as indicated by decreased renal neutrophil influx, but failed to show changes in intrarenal tumor necrosis factor-alpha production. Moreover, caspase-1-/- mice clearly exhibited reperfusion-induced apoptosis as reflected by renal terminal deoxynucleotidyltransferase histology and internucleosomal DNA cleavage. Treatment with IL-1 receptor antagonist, anti-IL-1 receptor antibody, or anti-IL-18 antibody minimally reduced renal functional deterioration, inflammation, and apoptosis. CONCLUSIONS: These findings suggest that activated caspase-1 and its inflammatory products are involved in, but not crucial to, the induction of inflammation after renal ischemia-reperfusion. Hence, apart from caspase-1, other (combinations of) activated caspases are likely to be more prominently involved in renal reperfusion injury.

Functional protection by acute phase proteins alpha(1)-acid glycoprotein and alpha(1)-antitrypsin against ischemia/reperfusion injury by preventing apoptosis and inflammation.

BACKGROUND: Ischemia followed by reperfusion (I/R) causes apoptosis, inflammation, and tissue damage leading to organ malfunction. Ischemic preconditioning can protect against such injury. This study investigates the contribution of the acute phase proteins alpha(1)-acid glycoprotein (AGP) and alpha(1)-antitrypsin (AAT) to the protective effect of ischemic preconditioning in the kidney. METHODS AND RESULTS: Exogenous AGP and AAT inhibited apoptosis and inflammation after 45 minutes of renal I/R in a murine model. AGP and AAT administered at reperfusion prevented apoptosis at 2 hours and 24 hours, as evaluated by the presence of internucleosomal DNA cleavage, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and the determination of renal caspase-1- and caspase-3-like activity. AGP and AAT exerted anti-inflammatory effects, as reflected by reduced renal tumor necrosis factor-alpha expression and neutrophil influx after 24 hours. In general, these agents improved renal function. Similar effects were observed when AGP and AAT were administered 2 hours after reperfusion but to a lesser extent and without functional improvement. Moreover, I/R elicited an acute phase response, as reflected by elevated serum AGP and serum amyloid P (SAP) levels after 24 hours, and increased hepatic acute phase protein mRNA levels after 18 hours of renal reperfusion. CONCLUSIONS: We propose that the antiapoptotic and anti-inflammatory effects of AGP and AAT contribute to the delayed type of protection associated with ischemic preconditioning and other insults. This mechanism is potentially involved in the course of many clinical conditions associated with I/R injury. Moreover, exogenous administration of these proteins may provide new therapeutic means of treatment.

Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model.

INTRODUCTION: Phosphatidylserine (PS) externalization is regarded as one of the earliest hallmarks of cells undergoing programmed cell death. We studied the use of labeled human recombinant annexin-V, a protein selectively binding to PS, to detect cardiomyocyte death in an in vivo mouse model of cardiac ischemia and reperfusion (I/R). METHODS AND RESULTS: I/R was induced in mouse hearts by ligation and subsequent release of a suture around the left anterior descending coronary artery. Annexin-V (25 mg/kg) fused to a marker molecule was injected intra-arterially 30 minutes before euthanasia. After 15 minutes of ischemia followed by 30 minutes of reperfusion, 1.4+/-1. 2% (mean+/-SD) of the cardiomyocytes in the area at risk were annexin-V positive (n=6). This increased to 11.4+/-1.9% after 15 minutes of ischemia followed by 90 minutes of reperfusion (n=7) and to 20.2+/-3.3% after 30 minutes of ischemia followed by 90 minutes of reperfusion (n=7). In control mice, including those injected with annexin-V at the binding site of PS, no annexin-V-positive cells were observed. DNA gel electrophoresis showed typical laddering starting after 15 minutes of ischemia followed by 30 minutes of reperfusion, suggesting activation of the cell death program. Intervention in the cell death program by pretreatment with a novel Na(+)-H(+) exchange inhibitor substantially decreased annexin-V-positive cardiomyocytes from 20.2% to 2.2% in mice after 30 minutes of ischemia followed by 90 minutes of reperfusion. CONCLUSIONS: These data suggest that labeled annexin-V is useful for in situ detection of cell death in an in vivo model of I/R in the heart and for the evaluation of cell death-blocking strategies.

Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation.

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

Insulin-like growth factor-1 (IGF-1) and growth hormone (GH) in immunity and inflammation.

In recent years many efforts have been undertaken to elucidate the complex interactions between mediators of the endocrine system and the immune system. The main effector of growth hormone (GH) is insulin-like growth factor-1 (IGF-1), an endocrine mediator of growth and development under physiological conditions. Besides this important function, IGF-1 also plays a prominent role in the regulation of immunity and inflammation. This article will address the involvement of IGF-1 in innate as well as acquired immunity and host-defense. We also discuss the role of IGF-1 in the course of inflammatory disorders, including sepsis and sepsis-induced catabolism as well as degenerative arthritis. Based on recent insights, we finally examine the pathophysiological background, potential pitfalls and perspectives of IGF-1 suppletion therapy in these conditions.

Ischemia/reperfusion-induced IFN-gamma up-regulation: involvement of IL-12 and IL-18.

Tissue injury as a consequence of ischemia followed by reperfusion is characterized by early as well as late signs of inflammation. The latter, among others, involves IFN-gamma-dependent up-regulation of MHC class I and II Ag expression. Employing a murine model of renal ischemia, we show that renal IL-18 mRNA up-regulation coincides with caspase-1 activation at day 1 following ischemia. IFN-gamma and IL-12 mRNA are subsequently up-regulated at day 6 following ischemia. Combined, but not separate, in vivo neutralization of the IFN-gamma inducing cytokines IL-12 and IL-18 reduces IFN-gamma-dependent MHC class I and II up-regulation to a similar extent as IFN-gamma neutralization, suggesting the involvement of functional IL-12, IL-18, and IFN-gamma protein. These results reveal a novel relationship between tissue injury of nonmicrobial origin and the induction of IL-12 as well as IL-18. The collaboration observed between endogenous IL-12 and IL-18 in the induction of IFN-gamma after renal ischemia/reperfusion, resembles the immune response to bacterial infections.

Involvement of endogenous interleukin-10 and tumor necrosis factor-alpha in renal ischemia-reperfusion injury.

BACKGROUND: Ischemia followed by reperfusion is a common clinical event associated with a pro-inflammatory response leading to organ dysfunction. The aim of the present study is to evaluate the interplay between this pro-inflammatory response and apoptosis. We investigated the role of the pro-inflammatory mediator tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory mediator interleukin-10 (IL-10) in inflammation and apoptosis after renal ischemia reperfusion. METHODS: Male Swiss mice were subjected to 45 min of ischemia followed by reperfusion and subsequently administered neutralizing Abs against either TNF-alpha (TN3), IL-10 (JES5-2A5) or control. RESULTS: After 1 day of reperfusion, anti-TNF-alpha treatment reduced whereas anti-IL-10 treatment exacerbated postischemic renal injury, inflammation, and, to a lesser extent, apoptosis as measured by changes in blood urea nitrogen content, immunohistologically detectable renal TNF-alpha protein and neutrophils, histological integrity of renal parenchyma, and DNA ladder formation. Furthermore, anti-IL-10 treatment enhanced major histocompatibility complex class I and II expression at day 7 as measured by enzyme immunoassay and immunohistology. CONCLUSIONS: These data indicate that the extent of reperfusion-induced apoptosis is modulated by the inflammatory response, during which locally produced TNF-alpha plays a significant role in the development of tissue injury. Subsequently, this pro-inflammatory reaction is followed by endogenous production of the anti-inflammatory cytokine IL-10, which serves as a physiological counterbalance to the effects of TNF-alpha. These novel pathophysiological insights may provide new basis for the development of tools for limiting ischemia and reperfusion injury.

Interference of pain control employing opioids in in vivo immunological experiments.

Pain control (PC) in laboratory animals is supported by ethical as well as methodological considerations, aimed at preventing an interfering reduction in food and water intake and normalizing stress hormone levels. However, little is known about the immunomodulatory attributes of analgesics, which putatively prevents the routine implementation of PC in immunological research. In an established murine model of endotoxemia we investigated the immunomodulatory properties of common clinical analgesics (the opioids fentanyl and buprenorphine). Additionally, a literature study was conducted to investigate the frequency of PC in laboratory animals used for immunological experimentation. In line with various reports, we observed interactions between the opioid analgesics and the immune system that altered the outcome of performed in vivo immunological experiments. Of 100 evaluated publications, none mentioned the use of PC, indicating its uncommon implementation. In conclusion, more studies on the interactions between the immune system and analgesics are needed to establish better criteria for adequate implementation. Finally, we propose that methodological sections in scientific journals should clearly document whether or not PC was employed. If PC is not used, the reason for not using it should be stated.

Motor denervation induces altered muscle fibre type densities and atrophy in a rat model of neuropathic pain.

Loose ligation of a sciatic nerve in rats (chronic constriction injury; CCI) provokes sensory, autonomic, and motor disturbances like those observed in humans with partial peripheral nerve injury. So far, it is unknown whether these motor disturbances result from (mechanical) allodynia or from damage to the motor neuron. These considerations prompted us to assess, in CCI rats, the density of motor axons in both the ligated sciatic nerve and the ipsilateral femoral nerve. To this end, we determined the number of cholinesterase positive fibres. It has been demonstrated previously that muscle fibre type density may be used as a measure of motor denervation and/or hypokinesia. Therefore, the myofibrillar ATPase reaction was employed to assess fibre type density in biopsies obtained from the lateral gastrocnemius muscle (innervated by sciatic nerve) and rectus femoris muscle (innervated by femoral nerve). We observed axonal degeneration of motor fibres within the loosely ligated sciatic nerve, both at an intermediate (day 21) and at a late stage (day 90) after nerve injury. The reduction in the number of motor nerve fibres was more pronounced distal to the site of the ligatures than proximal. A (less pronounced) reduction of motor fibres was observed in the ipsilateral (non-ligated) femoral nerve. In line with these findings, we observed altered fibre type densities in muscle tissue innervated by the ligated sciatic nerve as well as the non-ligated femoral nerve indicative of motor denervation rather than hypokinesia. The findings of this study suggest that the motor disorder induced by partial nerve injury involves degeneration of motor nerve fibres not only within the primarily affected nerve but also within adjacent large peripheral nerves. This spread outside the territory of the primarily affected nerve suggests degeneration of motor neurons at the level of the central nervous system.

Neurogenic inflammation in an animal model of neuropathic pain.

Loose ligation of a rat sciatic nerve (chronic constriction injury (CCI) model) provokes signs and symptoms like those observed in reflex sympathetic dystrophy (RSD) patients. Primary afferent nociceptive C-fibers seem to be involved in an afferent orthodromic as well as in an efferent antidromic manner. In this study we hypothesize that consequent to development of antidromic impulses in C-nociceptive afferents, neuropeptides released from peripheral endings of these fibers, increase skin blood flow (SBF), vascular permeability, and tissue accumulation of polymorphonuclear leukocytes (PMNs). Collectively, these phenomena have been referred to as neurogenic inflammation. To investigate the presence of neurogenic inflammation in the CCI-model, we assessed skin blood flow (SBF) as well as the level of edema and accumulation of PMNs in muscle tissue obtained from the affected hindpaw. SBF was measured, by means of laser Doppler flowmetry, before ligation as well as at day 4 after ligation. At day 4, SBF measurements were performed before and after abolition of the capability of C-fibers to mediate a vasodilator response. To this end, capsaicin was applied perineurally. Increased vascular permeability was inferred from the level of edema of muscle tissue as determined by assessment of wet/dry weight ratios of muscle biopsies. PMN accumulation was investigated by enzymatic detection of myeloperoxidase (MPO) activity in muscle biopsies. Compared with preligation values, at day 4 SBF was increased more than twofold (p < 0.05). The latter response was annihilated by capsaicin application. Compared with sham operated controls, wet/dry ratios were higher in the ligated animals (1.104 vs. 1.068; p < 0.05). Likewise, when compared with sham operated controls, MPO activity was found to be increased in the ligated hindpaw (Optic Density 0.15 vs. 0.89; p < 0.001). In conclusion, the findings of this study indicate that loose ligation of a sciatic nerve induces an inflammatory response in the ipsilateral hindpaw, which most likely is mediated by release of neuropeptides from the peripheral endings of antidromically acting nociceptive C-fibres.

Reflex sympathetic dystrophy: does sympathetic dysfunction originate from peripheral neuropathy?

BACKGROUND: Sympathetic dysfunction in reflex sympathetic dystrophy (RSD) has been purported to consist of an afferently-induced increase in efferent sympathetic nerve impulses (somato-sympathetic reflex) and/or denervation-induced supersensitivity to catecholamines. In addition, both the central and peripheral nervous systems have been claimed to be involved. It was the aim of this study to obtain more insights into these underlying mechanisms. METHODS: In the affected extremeties of 42 patients with RSD we investigated as indirect measures of sympathetic (dys)function: (1) skin blood flow and the vasoconstrictive response to dependency of skin microvessels by means of laser Doppler flowmetry (distal to the site of trauma), (2) relative distention of the brachial artery and changes in relative distention consequent to a cold pressor test by means of ultrasonic vessel wall tracking (proximal to the site of trauma), and (3) arterial blood pressures by means of the Finapres technique. Both provocation tests induce a sympathetically mediated response. Patients were divided into three categories according to their perception of skin temperature in their injured limb (stage I, stationary warmth sensation; stage II, intermittent warmth and cold sensation; or stage III, stationary cold sensation). RESULTS: Distal to the site of trauma, when compared with controls, skin blood flow was increased at stage I and decreased at stages II and III, whereas the vasoconstrictive response to dependency was impaired at all three stages. Proximally, when compared with controls, relative distention of the brachial artery and its response to the cold pressor test were decreased at all three stages. No differences were observed in pulse pressure between patient groups and controls. CONCLUSIONS: These results suggest that sympathetic dysfunction in extremities of patients with RSD distal to the site of trauma consists of hypersensitivity to catecholamines at stages II and III as a result of autonomic denervation at stage I, whereas proximal to the site of trauma sympathetic nerve impulses may be increased at all three stages.