Hypertonic saline and pentoxifylline reduces hemorrhagic shock resuscitation-induced pulmonary inflammation through attenuation of neutrophil degranulation and proinflammatory mediator synthesis

BACKGROUND: Ringer's lactate (RL), the current standard resuscitation fluid, potentiates neutrophil activation and is associated with pulmonary inflammation. Resuscitation with hypertonic saline and pentoxifylline (HSPTX) has been shown to attenuate hemorrhagic shock-induced injury when compared with RL. Because the neutrophil plays a major role in postshock inflammation, we hypothesized that HSPTX reduces pulmonary inflammation after resuscitation in comparison to RL. METHODS: Sprague-Dawley rats underwent controlled shock and were resuscitated with RL (32 mL/kg) or HSPTX (4 mL/kg 7.5% NaCl + pentoxifylline 25 mg/kg). Animals who did not undergo shock or resuscitation served as controls. After 24 hours, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. Cytokine induced neutrophil chemoattractant (CINC) was measured in BALF by enzyme-linked immunosorbent assay. Matrix metalloproteinases (MMP)-2 and -9 were measured by zymography. Hemeoxygenase-1 (HO-1) was assessed by Western blot and immunohistochemistry. RESULTS: HSPTX resuscitation led to a 62% decrease in CINC levels compared with RL (p < 0.01). BALF MMP-2 expression was attenuated by 11% with HSPTX (p = 0.09). Lung MMP-2 and MMP-9 expression was reduced by 89% (p < 0.01) and 76%, respectively (p < 0.05). Lung HO-1 expression declined by 34% with HSPTX in comparison to RL (p < 0.01), indicating less oxidative injury. Lung immunohistochemistry localized HO-1 to neutrophils, macrophages, and airway epithelial cells. CONCLUSION: Collectively, the attenuation of pulmonary inflammation with HSPTX after shock when compared with RL is associated with downregulation of neutrophil activation, oxidative stress, and proinflammatory mediator production.     

Fracture hematoma is a potent proinflammatory mediator of neutrophil function

BACKGROUND: Patients with multiple skeletal injuries are susceptible to acute respiratory distress syndrome and multiple organ failure, which result from hyperactivation of the immune system. This study was designed to evaluate in vitro the proinflammatory properties of fracture hematoma (FH). METHODS: FH was isolated from patients undergoing emergent open reduction and internal fixation for isolated closed fractures. Neutrophils (PMNs), isolated from healthy volunteers, were exposed to the FH supernatant and activation was examined (CD11b and CD18 adhesion receptor expression and respiratory burst). PMN phagocytosis, apoptosis, and transmigration across an endothelial barrier were also assessed. RESULTS: FH increased PMN respiratory burst (control, 100; FH-treated, 186) and phagocytosis (control, 100; FH-treated, 172) but had no effect on adhesion receptor expression. Transendothelial migration of PMNs was unaffected, although FH was toxic to endothelial cells. In contrast, apoptosis of FH-treated PMNs was delayed (control, 46; FH-treated, 8). CONCLUSION: These effects, although beneficial at the site of injury in the context of antibactericidal function, may cause PMN-mediated tissue injury systemically.     

Pancreatic somatostatin is a mediator of glucagon inhibition by hyperglycemia

We have previously shown that a nonimmunoreactive analogue of somatostatin, (D-Ala5, D-Trp8)-somatostatin, differentially inhibits pancreatic somatostatin secretion without inhibiting insulin or glucagon secretion. During normoglycemia, suppression of pancreatic somatostatin with this analogue increases glucagon and insulin secretion, suggesting that pancreatic somatostatin tonically inhibits glucagon and insulin secretion by a paracrine mechanism. In our study, we used this analogue to determine whether endogenous pancreatic somatostatin has a role in the inhibition of glucagon secretion by hyperglycemia. The experiments were performed in pentobarbital-anesthetized, laparotomized dogs. To measure the pancreatic output of somatostatin directly, pancreatic venous blood was sampled from the right lobe of the dog pancreas, and the pancreatic blood flow was measured. In the first set of experiments, glucagon secretion was suppressed by a glucose infusion (200 mg/kg bolus and 20 mg X kg-1 X min-1 i.v.) for 3 h. Plasma glucose rose from 102 +/- 6 to 365 +/- 34 mg/dl. Pancreatic insulin output increased 10-fold, pancreatic somatostatin output increased from 1.2 +/- 0.3 to 3.0 +/- 0.8 ng/min, and pancreatic glucagon output was suppressed from 1.4 +/- 0.7 to 0.5 +/- 0.1 ng/min. After 2 h of glucose infusion, an infusion of the analogue (5.5 micrograms/min i.v.) reversed both the stimulation of somatostatin and the suppression of glucagon without significantly changing either the plasma glucose level or the pancreatic insulin output. In a second set of experiments, basal somatostatin output was suppressed by the analogue (5.5 micrograms/min i.v.) for 15 min before the administration of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ischemia-reperfusion induces glomerular and tubular activation of proinflammatory and antiapoptotic pathways: differential modulation by rapamycin

Ischemia-reperfusion (I-R) injury in transplanted kidney, a key pathogenic event of delayed graft function (DGF), is characterized by tubular cell apoptosis and interstitial inflammation. Akt-mammalian target of rapamycin-S6k and NF-kappaB-inducing kinase (NIK)-NF-kappaB axis are the two main signaling pathways regulating cell survival and inflammation. Rapamycin, an immunosuppressive drug inhibiting the Akt axis, is associated with a prolonged DGF. The aim of this study was to evaluate Akt and NF-kappaB axis activation in patients who had DGF and received or not rapamycin and in a pig model of I-R and the role of coagulation priming in this setting. In graft biopsies from patients who were not receiving rapamycin, phosphorylated Akt increased in proximal tubular, interstitial, and mesangial cells with a clear nuclear translocation. The same pattern of activation was observed for S6k and NIK. However, in rapamycin-treated patients, a significant reduction of S6k but not Akt and NIK activation was observed. A time-dependent activation of phosphatidylinositol 3-kinase, Akt, S6k, and NIK was observed in the experimental model with the same pattern reported for transplant recipients who did not receive rapamycin. Extensive interstitial and glomerular fibrin deposition was observed both in pig kidneys upon reperfusion and in DGF human biopsies. It is interesting that the activation of both Akt and NIK-NF-kappaB pathways was induced by thrombin in cultured proximal tubular cells. In conclusion, the data suggest that (1) coagulation may play a pathogenic role in I-R injury; (2) the Akt axis is activated after I-R, and its inhibition may explain the prolonged DGF observed in rapamycin-treated patients; and (3) NIK activation in I-R and DGF represents a proinflammatory, rapamycin-insensitive signal, potentially leading to progressive graft injury.     

Enhancement of bone morphogenetic protein-2-induced new bone formation in mice by the phosphodiesterase inhibitor pentoxifylline

Porous collagen disks (6 mm diameter, 1 mm thickness) were impregnated with recombinant human bone morphogenetic protein-2 (rhBMP-2) (5 microg/disk) and implanted onto the back muscles of mice. Pentoxifylline (PTX), which is a methylxanthine-derived inhibitor of phosphodiesterases (PDEs), or vehicle, was injected (5, 25, 50, 100, 200, and 300 mg/kg body weight/day) into the mice subcutaneously once a day for 3 weeks from the day of implantation of the bone morphogenetic protein (BMP)-laden disks. The rhBMP-2-induced ectopic ossicles were harvested and examined using radiographic, histological, and biochemical methods to determine size, bone quality, and calcium content. When compared with controls, ossicles from mice treated with >50 mg/kg per day of PTX were significantly larger in size and had a greater calcium content. However, no differences were noted in mice treated with lower doses (5 and 25 mg/kg per day) of PTX. The temporal sequence of the bone-forming process was unchanged by PTX based on histological examination. The histology of the ossicles from high- and low-dose PTX-treated mice was essentially identical to that observed in the control mice. These experimental results indicate that PTX enhanced the bone-inducing capacity of BMP-2. The underlying mechanism of action most likely involves the inhibition of intracellular phosphodiesterases and a resulting elevation of the intracellular content of cyclic nucleotides. Further studies are warranted to understand how BMP-induced bone formation is pharmacologically modified by PTX.     

Neutrophil degranulation and the effects of phosphodiesterase inhibition

BACKGROUND: Neutrophils play a major role as the first line in host defense after exposure to bacterial products. However, an exaggerated inflammatory response characterized by overwhelming neutrophil activation can be injurious to the host. Pentoxifylline (PTX), a nonspecific phosphodiesterase inhibitor, has been shown to attenuate neutrophil oxidative burst and decrease proinflammatory mediator synthesis. We hypothesized that PTX down-regulates neutrophil activation by decreasing the surface expression of both CD35 and CD66b, two markers of neutrophil degranulation. MATERIALS AND METHODS: Venous blood was obtained from three healthy volunteers. Whole blood was incubated with HBSS (control), f-methionyl-leucyl-phenylalanine (fMLP, 1 microM/L), PTX (2 mM/L), or fMLP + PTX. CD35 and CD66b expression were measured by flow cytometry. RESULTS: fMLP treatment caused a significant increase in CD35 and CD66b expression of when compared to controls (P < 0.01). PTX treatment revealed expression of both markers comparable to the control group. A 38% decrease in CD35 (64 +/- 12 versus 100; P < 0.01) and a 52% decrease in CD66b (48 +/- 7 versus 100; P < 0.01) expression were demonstrated in the fMLP + PTX group when compared to fMLP alone. CONCLUSION: In addition to the known effects of PTX on neutrophil oxidative burst, PTX also affects neutrophil degranulation, an essential step in enzyme release and subsequent tissue injury. These findings may have clinical relevance in the treatment of disease processes due to inflammation in which primed neutrophils play a role.     

Mechanism of the beneficial effects of pentoxifylline during sepsis: maintenance of adrenomedullin responsiveness and downregulation of proinflammatory cytokines

BACKGROUND: Although it is known that pentoxifylline (PTX) produces various beneficial effects during sepsis, it remains unknown whether this agent has any salutary effects on the depressed vascular responsiveness to adrenomedullin (ADM), a novel potent vasodilatory peptide, under such conditions. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). One hour after CLP, PTX (50 mg/kg body wt) or vehicle (normal saline) was infused intravenously over 90 min. Twenty hours after CLP (i.e., the late, hypodynamic stage of sepsis), the thoracic aorta and small intestine were isolated and preconstricted by norepinephrine. Rat ADM (10(-7) M) was applied, and the percentage of ADM-induced relaxation in the aortic rings and resistance vessels in the small intestine was determined. In addition, plasma ADM was determined by radioimmunoassay and tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 levels were measured by enzyme-linked immunosorbent assay. RESULTS: The percentage of ADM-induced vascular relaxation in the aortic rings and resistance vessels of the isolated gut was significantly reduced 20 h after CLP. Administration of PTX early after the onset of sepsis, however, prevented the decrease in vascular ADM responsiveness at the macro- and microcirculatory levels. Plasma ADM levels increased after CLP, irrespective of PTX infusion, indicating that the effect of PTX was not mediated by altering ADM release. The upregulated TNF-alpha, IL-1beta, and IL-6 during late sepsis were, however, attenuated by PTX administration, suggesting that maintenance of ADM responsiveness by this agent appears to be due to downregulation of these cytokines. CONCLUSIONS: Since early administration of PTX maintains vascular ADM responsiveness even during the late stage of sepsis, this agent appears to be a useful adjunct in preventing the deterioration in hemodynamics and cardiovascular function during the progression of polymicrobial sepsis.     

Suppression of PMN apoptosis by hypoxia is dependent on Mcl-1 and MAPK activity

BACKGROUND: Hypoxia has been shown to delay the onset of neutrophil (polymorphonuclear leukocytes [PMNs]) apoptosis. With the use of antisense oligonucleotides, we have previously demonstrated that Mcl-1 is necessary for this effect. We wanted to further characterize the expression of Mcl-1 and examine signaling pathways required for the delay in apoptosis that is mediated by hypoxia. METHODS: For kinase signaling inhibition, PMNs were incubated for 12 hours with the following inhibitors: PD98059 Mitogen Activated Protein Kinase Kinase (MEK), SB202190 (p38 mitogen-activated protein kinase [MAPK]), and LY294002 (phosphatidyl inositol-3-kinase [PI3K]). PMNs that were treated with inhibitors were assessed for apoptosis by morphologic features or were lysed for Western blot analysis. RESULTS: Western blot analyses, immunofluorescent staining, and quantification showed an upregulation of Mcl-1 expression after 12 hours of incubation in response to hypoxia. When inhibitors of either MEK or p38 MAPK were incubated with PMNs during hypoxia, apoptosis increased to similar levels as normoxia. We further wanted to determine whether signaling through p38 MAPK or MEK led to increased Mcl-1 expression. Western blot analysis confirmed that the inhibition of p38 MAPK led to a significant decrease in Mcl-1 expression. CONCLUSIONS: We have documented a novel mechanism by which hypoxia can modify PMN apoptosis in the wound site by the activation of p38 MAPK signaling, thereby inducing the anti-apoptotic protein Mcl-1.     

补体在重度烧伤病人PMN吞杀绿脓杆菌力中的作用

采集了７０例烧伤病人１２９份外周血中性粒细胞（ＰＭＮ），以Ｏ~２￣－、特殊颗粒（ＳＧ）和胞内杀菌力（ＩＣＢＡ）为指标动态观测了深度烧伤后病人的ＰＭＮ水平变化；分析了病人血浆对正常人ＰＭＮ胞内指标的有害作用，及抗人Ｃ~３、Ｃ~５血清（ＡＨＣ~３Ｃ~５Ｓ）对上述有害作用的特异阻断效能。结果表明：①重、中度烧伤病人的各指标值均显著低于正常水平（Ｐ＜０．０５）；当ＢＳＤ（深度烧伤面积）≥４５％和病程在１～６天时，ＩＣＢＡ最低。②ＩＣＢＡ与ＳＧ和Ｏ~２￣－的动态相关显著（Ｐ＜０．０５）。③病人血浆能显著削减正常人ＰＭＮ胞内ＩＣＢＡ、ＳＧ和Ｏ~２￣－的储存，而ＡＨＣ~３Ｃ~５Ｓ能减轻这些储存的丢失，效能由保护率表示：ＩＣＢＡ（６７．３３％）＞ＳＧ（５１．６０％）＞Ｏ~２￣－（４６．６８％）。结果提示：ＰＭＮ杀菌力的高低与ＢＳＤ和病程早晚呈负相关，Ｃ~３、Ｃ~５碎片是ＰＭＮ胞内ＩＣＢＡ下降的重要直接因素。     

补体在重度烧伤病人PMN吞杀绿脓杆菌力中的作用

采集了70例烧伤病人129份外周血中性粒细胞(PMN),以O_2、特殊颗粒(SG)和胞内杀菌力(ICBA)为指标动态观测了深度烧伤后病人的PMN水平变化;分析了病人血浆对正常人PMN胞内指标的有害作用,及抗人C_3、C_3血清(AHC_3C_3S)对上述有害作用的特异阻断效能。结果表明:①重、中度烧伤病人的各指标值均显著低于正常水平(P<0.05);当BSD(深度烧伤面积)≥45％和病程在1～6天时,ICBA最低。②1CBA与SG和O_2的动态相关显著(P<0.05)。③病人血浆能显著削减正常人PMN胞内1CBA、SG和O_2~-的储存,而AHC_3C_S能减轻这些储存的丢失,效能由保护率表示;ICBA(67.33％)>SG(51.60％)>O_2~-(46.68％)。结果提示:PMN杀菌力的高低与BSD和病程早晚呈负相关,C_3、C_5碎片是PMN胞内ICBA下降的重要直接因素。     

烧伤后白细胞与内皮细胞粘附分子介导作用的研究

为研究白细胞粘附依赖于细胞粘附分子的介导作用,作者通过离体实验观察分析了烧伤血清对外周血中性粒细胞（ＰＭＮ）ＣＤ１１ｂ／ＣＤ１８表达的影响及ＣＤ１１ｂ／ＣＤ１８在烧伤后ＰＭＮ粘附中的介导作用。结果表明：（１）烧伤血清使ＰＭＮＣＤ１１ｂ／ＣＤ１８分子表达和ＰＭＮ与肺微血管内皮细胞（ＰＭＥＣ）的粘附率增高。（２）ＣＤ１１ｂ／ＣＤ１８单抗不仅能使正常ＰＭＮ与ＰＭＥＣ的粘附率下降５０％,也使烧伤血清激活的ＰＭＮ与ＰＭＥＣ粘附率下降至烧伤血清激活前水平。我们认为,严重烧伤能使外周血ＰＭＮＣＤ１１ｂ／ＣＤ１８表达明显增高,ＣＤ１１ｂ／ＣＤ１８不但介导基础状态下的ＰＭＮ粘附,更是介导烧伤后引起的大量ＰＭＮ与ＰＭＥＣ粘附的主要分子。     

Nitric oxide inhibition of IGF-1 stimulated proteoglycan synthesis: role of cGMP.

Insulin-like growth factor (IGF-1) is critical for normal development and maintenance of cartilage, however arthritic cartilage responds poorly to IGF-1; part of this insensitivity is mediated by nitric oxide (NO). These studies test if cGMP is responsible for NO dependent insensitivity to IGF-1 in chondrocytes in situ in organ culture and in monolayer culture. Lapine cartilage and chondrocytes in monolayer culture and cartilage from osteoarthritic human knees were used. Tissues were exposed to NO from iNOS induced by IL-1, and proteoglycan synthesis in response to IGF-1 was evaluated in the presence and absence of cGMP dependent protein kinase (PKG) inhibitors. PKG activators inhibited IGF-1 responses in cartilage but not chondrocytes in monolayer. IL-1 stimulated cGMP synthesis in both monolayer and organ cultures. However, PKG inhibitors in cartilage slices but not in monolayer cultures restored response to IGF-1. PKG activity was detected in both fresh and monolayer chondrocytes, confirming this part of the cGMP signal cascade is intact in both of the preparations evaluated. Arthritic cartilage response to IGF-1 was restored by both N(G)-monomethyl-L-arginine inhibition of NO synthesis and PKG inhibitors. The data suggests that cGMP mediated effects are critical to NO actions on chondrocytes in situ in the cartilage matrix and supports a role for cGMP in the pathophysiologic effects of NO in osteoarthritis.     

Complement activation by the alternative pathway is modified in renal failure: the role of factor D

Factor D, an essential enzyme of the alternative pathway (AP) of complement, is eliminated by the kidney, and its plasma concentration increases 10-fold in end-stage renal disease (ESRD). The purpose of this study was to analyze the consequences of factor D accumulation. A number of in vitro assays were used to analyze AP activation in normal human serum (NHS), in normal serum supplemented with purified factor D to 10-fold its normal concentration (10 x D), and in sera of patients with ESRD. When compared with NHS, in 10 x D: 1) Spontaneous fluid-phase activation of complement at 37 degrees C was greatly increased as measured by C3 cleavage, 2) The lysis of rabbit erythrocytes, a function of the AP, was accelerated, 3) More C3 fragments bound to cuprophane membranes and to immune precipitates; both reactions were accompanied by the formation of more C5a, 4) Complement mediated solubilization of antigen-antibody precipitates was enhanced. Sera of patients with ESRD behaved similarly to 10 x D in all assays used, i.e., enhanced AP function, although complement activation measured in these assays varied widely from one individual to another. Thus, the elevated factor D concentration observed in renal failure might have important pathophysiological consequences, some of which could be detrimental (e.g., C5a produced during hemodialysis), while others might be beneficial, e.g., solubilization of immune precipitates.