Mast cells modulate the inflammatory but not the proliferative response in healing wounds

Upon stimulation, mast cells release a heterogeneous group of factors that promote inflammation and influence cell proliferation. Mast cells accumulate at sites of injury, further suggesting a critical role in wound healing. To assess the importance of mast cells in tissue repair, we compared wound healing in mast cell-deficient WBB6F1/J-KitW/KitW-v (KitW/KitW-v) and wild type WBB6F1/++ (WT) mice. During the inflammatory phase, neutrophil infiltration into wounds of the KitW/KitW-v mice was significantly less than that of WT mice (84.6 +/- 10.3 vs. 218 +/- 26.0 cells/10 high-power fields at day 3, p < 0.001), while wound macrophage and T cell infiltration were similar in both strains. The decrease in neutrophils could not be explained by changes in tumor necrosis factor-alpha or macrophage inflammatory protein-2 levels, because the amounts of these two neutrophil chemoattractants were similar in both KitW/KitW-v and WT mice. Surprisingly, the absence of mast cells had no effect on the proliferative aspects of wound healing, including reepithelialization, collagen synthesis, and angiogenesis. Although mast cells are known to release proangiogenic mediators, vascular endothelial growth factor levels were similar in WT and KitW/KitW-v mice. Moreover, levels of fibroblast growth factor-2 were increased in KitW/KitW-v mice (4206 +/- 107 vs. 1865 +/- 249 pg/ml, p < 0.01). These results suggest that mast cells modulate the recruitment of neutrophils into sites of injury, yet indicate that mast cells are unlikely to exert a major influence on the proliferative response within healing wounds.     

CD44-mediated neutrophil apoptosis in the rat

BACKGROUND: Apoptosis is an important mechanism by which neutrophils are removed from sites of inflammation, including the kidney. This study investigated whether ligation of the cell-surface adhesion molecule, CD44, can trigger neutrophil apoptosis. METHODS: The anti-rat CD44 antibody OX-50 was used to induce apoptosis of cultured blood neutrophils, as determined by flow cytometry using annexin V staining and by transmission electron microscopy. The functional consequences of OX-50-mediated neutrophil depletion were examined in a rat model of accelerated antiglomerular basement membrane glomerulonephritis. RESULTS: Flow cytometric analysis using the OX-50 antibody, which recognizes the common amino terminal domain of CD44, showed that rat blood neutrophils express very high levels of CD44. The addition of OX-50, but not control antibodies, rapidly induced neutrophil apoptosis in cultured rat blood leukocytes, as demonstrated by annexin V staining and by electron microscopy. Cross-linking of CD44 was essential since F(ab) fragments of the OX-50 antibody failed to induce neutrophil apoptosis. The CD44 ligand hyaluronan and an antibody to the CD44v6 isoform failed to induce neutrophil apoptosis, indicating that OX-50 antibody-mediated neutrophil apoptosis is epitope specific. This effect was specific to neutrophils since the OX-50 antibody did not induce apoptosis in other CD44-expressing cell types (lymphocytes, mesangial cells, or tubular epithelial cells). An injection of OX-50 antibody into normal rats caused a rapid and profound neutropenia, and apoptotic neutrophils could be seen in the blood by electron microscopy. Furthermore, the administration of OX-50 antibody abrogated neutrophil-dependent glomerular injury (proteinuria) on day 1 of rat antiglomerular basement membrane glomerulonephritis, whereas injury on day 10 of the disease (neutrophil independent) was largely unaffected. CONCLUSIONS: The cross-linking of specific epitopes of the CD44 molecule can rapidly induce neutrophil apoptosis in vitro and inhibit neutrophil-dependent renal injury in vivo. This finding suggests that physiological ligands of the CD44 molecule may play an important role in eliminating neutrophils from sites of inflammation, including inflammatory kidney disease.     

Differential effects of steroids on leukocyte-mediated glomerulonephritis in the rabbit

The effects of steroids on the development of injury in two models of experimental glomerulonephritis (GN), (one mediated by neutrophils, the other by macrophages) were compared. The neutrophil-associated lesion [initiated by heterologous antiglomerular basement membrane (GBM) antibody] was characterized by the development of an exudative endocapillary GN with heavy neutrophil accumulation [mean, 6.9 neutrophils/glomerular cross section (N/GCS) +/- 2.9 SD], minor macrophage infiltration [7.9 macrophages/glomerulus (M/G) +/- 2.2 SD] and heavy proteinuria (1905 mg/24 hr +/- 520 SD). Steroid-treated (methylprednisolone, 2 mg/kg/12 hr i.v.) rabbits developed a marked monocytopenia, mild neutrophilia, and significant reduction in glomerular macrophage accumulation (0.3 M/G 0.02 SD). However, neutrophil accumulation (6.1 N/CGS +/- 2.5 SD), histological appearances, and proteinuria (1820 mg/hr +/- 490 SD) were unaffected. The macrophage-associated model of GN was induced by passive autologous rabbit anti-sheep IgG 15 hr after the injection of a subnephritogenic dose of the same anti-GBM antibody. The glomerular lesion was characterized by a diffuse endocapillary proliferative GN with heavy macrophage infiltration (54 M/G +/- 8 SD), insignificant neutrophil accumulation (0.8 N/GCS 0.02 SD), and the regular development of proteinuria (420 mg/24 hr +/- 80 SD). Steroid-treated rabbits developed a mild neutrophilia and a significant monocytopenia associated with abrogation of glomerular macrophage accumulation (2.3 M/G +/- 0.8 SD). This was associated with the prevention of the development of GN and proteinuria (22 +/- 9.5 SD). Thus, steroids produce monocytopenia and prevent glomerular macrophage accumulation and associated injury whereas neutrophil accumulation and injury is unaffected. These data suggest steroids may have widely varying effects on the outcome of leukocyte-associated experimental GN depending on the nature of the infiltrating cells.     

Inhibition of neutrophil apoptosis after elective surgery.

BACKGROUND: Neutrophils play a crucial role in host defense against infections, but their inappropriate infiltration and activation within tissues can cause host tissue damage through release of reactive oxygen metabolites, metalloproteinases, and proinflammatory cytokines. The termination of a neutrophil-mediated inflammatory response is effected through programmed cell death or apoptosis. Delayed neutrophil apoptosis is associated with proinflammatory diseases, such as the systemic inflammatory response syndrome. Surgery induces a profound inflammatory response; therefore, neutrophil apoptosis of patients undergoing elective surgery was investigated. METHODS: Nonseptic patients undergoing elective orthopedic surgery while under epidural anesthesia had neutrophils and platelet-poor isolated from whole venous blood harvested at 4 time points: pre-epidural, 45 minutes postepidural but before surgical intervention, 1 hour postsurgical incision, and 24 hours postsurgery. Neutrophil apoptosis was quantified at 1, 12, and 24 hours in culture by immunofluorescence flow cytometry of annexin V and propidium iodide staining and confirmed by TUNEL (terminal deoxynucleotidyl transferase nick end labeling) assay for DNA strand breaks. Serum cytokines were quantified by specific enzyme-linked immunosorbent assay. RESULTS: Spontaneous neutrophil apoptosis after elective surgery was significantly (P < .001) inhibited with an effect evident within an hour of surgical incision and persisting at 24 hours postsurgery. The addition of patients' 24 hour postoperative plasma to healthy neutrophils markedly (P < .01) reduced neutrophil apoptosis, whereas plasma taken an hour after surgical incision was ineffective. Interleukin (IL)-6 was notably increased (1395 +/- 196 pg/mL, P < .01) 24 hours postsurgery and at this postoperative concentration inhibited (P < .01) apoptosis of normal neutrophils. Levels of other inflammatory mediators (IL-1 beta, tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, soluble Fas, soluble Fas ligand) were unaltered. The anti-inflammatory cytokine IL-10 was only slightly increased 24 hours postsurgery (8.32 +/- 2.99 pg/mL); however, the addition of recombinant human IL-10 (10 ng/mL) counteracted (P < .05) inhibition of neutrophil apoptosis induced by IL-6 and post-surgery plasma. CONCLUSIONS: These results identify marked inhibition of neutrophil apoptosis after elective surgery and suggest that the inhibition of neutrophil apoptosis in the postoperative period is, at least in part, a result of soluble circulating factors. The marked imbalance favoring proinflammatory over anti-inflammatory cytokine release in the immediate postoperative period mediates the overwhelmingly antiapoptotic net capacity of postsurgery plasma.     

Isoflurane inhibits cardiac myocyte apoptosis during oxidative and inflammatory stress by activating Akt and enhancing Bcl-2 expression

BACKGROUND: Volatile anesthetics attenuate apoptosis. The underlying mechanisms remain undefined. The authors tested whether isoflurane reduces apoptosis in cardiomyocytes subjected to oxidative or inflammatory stress by enhancing Akt and B-cell lymphoma-2 (Bcl-2). METHODS: Adult and neonatal rat ventricular myocytes and atrial HL-1 myocytes were exposed to hypoxia, hydrogen peroxide, or neutrophils with or without isoflurane pretreatment. The authors assessed cell damage and investigated apoptosis using mitochondrial cytochrome c release, caspase activity, and TUNEL assay. They determined expression of phospho-Akt and Bcl-2 and tested their involvement by blocking phospho-Akt with wortmannin and Bcl-2 with HA14-1. RESULTS: Isoflurane significantly reduced the cell damage and apoptosis induced by hypoxia, H2O2, and neutrophils. Isoflurane reduced hypoxia-induced mitochondrial cytochrome c release in HL-1 cells by 45 +/- 12% and caspase activity by 28 +/- 4%; in neonatal cells, it reduced caspase activity by 43 +/- 5% and TUNEL-positive cells by 50 +/- 2%. Isoflurane attenuated H2O2-induced caspase activity in HL-1 cells by 48 +/- 16% and TUNEL-positive cells by 78 +/- 3%; in neonatal cells, it reduced caspase activity by 30 +/- 3% and TUNEL-positive cells by 32 +/- 7%. In adult cardiomyocytes exposed to neutrophils, isoflurane decreased both mitochondrial cytochrome c and caspase activity by 47 +/- 3% and TUNEL-positive cells by 25 +/- 4%. Isoflurane enhanced phospho-Akt and Bcl-2 expression. Wortmannin and HA14-1 prevented the action of isoflurane (53 +/- 8% and 54 +/- 7% apoptotic cells vs. 18 +/- 1% without blockers). CONCLUSIONS: Isoflurane protects cardiomyocytes against apoptosis induced by hypoxia, H2O2, or activated neutrophils through Akt activation and increased Bcl-2 expression. This suggests that a reduction in apoptosis contributes to the cardioprotective effects of isoflurane.     

Isoflurane Inhibits Cardiac Myocyte Apoptosis during Oxidative and Inflammatory Stress by Activating Akt and Enhancing Bcl-2 Expression

Background: Volatile anesthetics attenuate apoptosis. The underlying mechanisms remain undefined. The authors tested whether isoflurane reduces apoptosis in cardiomyocytes subjected to oxidative or inflammatory stress by enhancing Akt and B-cell lymphoma-2 (Bcl-2).

Methods: Adult and neonatal rat ventricular myocytes and atrial HL-1 myocytes were exposed to hypoxia, hydrogen peroxide, or neutrophils with or without isoflurane pretreatment. The authors assessed cell damage and investigated apoptosis using mitochondrial cytochrome c release, caspase activity, and TUNEL assay. They determined expression of phospho-Akt and Bcl-2 and tested their involvement by blocking phospho-Akt with wortmannin and Bcl-2 with HA14-1.

Results: Isoflurane significantly reduced the cell damage and apoptosis induced by hypoxia, H2O2, and neutrophils. Isoflurane reduced hypoxia-induced mitochondrial cytochrome c release in HL-1 cells by 45 +/- 12% and caspase activity by 28 +/- 4%; in neonatal cells, it reduced caspase activity by 43 +/- 5% and TUNEL-positive cells by 50 +/- 2%. Isoflurane attenuated H2O2-induced caspase activity in HL-1 cells by 48 +/- 16% and TUNEL-positive cells by 78 +/- 3%; in neonatal cells, it reduced caspase activity by 30 +/- 3% and TUNEL-positive cells by 32 +/- 7%. In adult cardiomyocytes exposed to neutrophils, isoflurane decreased both mitochondrial cytochrome c and caspase activity by 47 +/- 3% and TUNEL-positive cells by 25 +/- 4%. Isoflurane enhanced phospho-Akt and Bcl-2 expression. Wortmannin and HA14-1 prevented the action of isoflurane (53 +/- 8% and 54 +/- 7% apoptotic cells vs. 18 +/- 1% without blockers).     

Future directions in the treatment of IgA nephropathy

IgA nephropathy (IgAN) is the most common primary glomerulonephritis yet its etiology remains uncertain. Recent data suggest a structural aberration of the IgA molecule in IgAN that may exert pathophysiologic effects on target cells, reduce clearance of IgA-immune complexes (IC), or favor mesangial IC trapping. Mesangial reactivity to immune complexes triggers off the release of cytokines and the alteration of prostaglandin and thromboxane A(2) production promoting mesangial cell proliferation. Angiotensin II-induced mesangial cells contraction with efferent arteriolar vasodilatation initiates glomerular injury and eventually lead to glomerulosclerosis following increased local production of transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF). This paper highlights the potential therapeutic strategies in the future. These strategies include: (i) decreasing the synthesis of IgA-IC; (ii) limiting the mesangial uptake of IgA-IC; (iii) antagonizing the effect of PDGF and TGF-beta to reduce mesangial proliferation and glomerulosclerosis; and (iv) reducing the noxious glomerular injury due to infiltrating neutrophils. The effective treatment of IgAN requires a better clarification of the pathogenesis of the nephropathy. Future therapeutic attempts to slow down the renal deterioration should target at prevention of mesangial IgA deposition and the amelioration of inflammatory injury induced by infiltrating neutrophils and the released cytokines.     

In vivo comparison of the inflammatory response induced by different vascular biomaterials

Biomaterial implants induce a local inflammatory response. A comparison of the inflammatory cell response was made between several biomaterials commonly used as vascular prostheses. Disks of polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), aluminum, titanium, copper, and stainless steel were surgically placed into the peritoneum of mice. Recruited macrophage and neutrophil populations were measured after recovery from the disk surface and peritoneal lavage. Following peritoneal biomaterial implants, there was no difference in total neutrophil or macrophage recruitment between mice implanted with PET, PTFE, aluminum, or titanium disks. However, there was significant attenuation of total neutrophil and macrophage recruitment to stainless steel compared with the other implants. Similarly, there was no significant difference in the percentage of leukocytes adherent to the PET, aluminum, or titanium disks. Macrophage adherence to the stainless steel disks was attenuated by 19.1%, and the number of neutrophils was attenuated by 69.1% when compared with PET implant mice. Mice implanted with copper disks universally expired. Leukocyte recruitment did not differ between PET, PTFE, aluminum, or titanium disks, suggesting that these materials stimulate similar inflammatory responses. Stainless steel disks recruited both fewer neutrophils and fewer macrophages and support lower adherence of these cells than the other biomaterials. Copper incited an overwhelming and fatal response.     

Acceleration of neutrophil apoptosis by glucose-containing peritoneal dialysis solutions: role of caspases.

Commercial, glucose-containing peritoneal dialysis (PD) solutions have deleterious effects on leukocytes and mesothelial cells that contribute to an impaired peritoneal defense. However, the molecular mechanisms of these deleterious effects are poorly understood. The effect of PD solutions on neutrophil viability, the molecular mechanisms of cell death, its functional consequences, and the possibilities for pharmacologic modulation have now been studied. The effect of newly available, bicarbonate-buffered PD solutions were further investigated. Lactate-buffered, glucose-containing PD solutions increased the apoptosis rate of cultured neutrophils (control media versus 4.25% glucose PD solution: 31 +/- 3% versus 52 +/- 3% apoptosis at 24 h, P < 0.001). Bicarbonate-buffered, 4.25% glucose-containing PD solutions with low concentration of glucose degradation products did not increase the rate of apoptosis. Apoptosis induced by lactate-buffered, 4.25% glucose PD solutions was not related to hyperosmolality or acidic pH and was not reproduced by increasing the glucose concentration by the addition of glucose to a commercial, lactate-buffered fluid. Neutrophil apoptosis was associated with caspase-3 activation. Inhibition of caspase-3 by the use of the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-fmk or the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) prevented features of apoptosis, such as morphologic changes, internucleosomal DNA degradation, and the appearance of hypodiploid cells and increased the number of viable, trypan blue-excluding neutrophils. Furthermore, zVAD-fmk increased neutrophil phagocytosis of bacteria. However, the caspase-1 inhibitor acetyl-Tyr-Val-Ala-Asp-aldehyde did not prevent cell death. These data suggest that unidentified components in commercial, lactate-buffered, high-glucose PD fluid accelerate the rate of neutrophil apoptosis. Glucose degradation products may be such unidentified components. Acceleration of neutrophil apoptosis may contribute to the impaired local defense system of patients undergoing PD.     

Suppressed neutrophil oxidative activity in sepsis: a receptor-mediated regulatory response

We have reported that neutrophils from patients with intraabdominal sepsis show suppressed superoxide production in response to soluble chemoattractants. To determine whether this could be a result of altered receptor function, neutrophil function studies and characterization of receptors were performed upon 22 patients recently operated upon for intraabdominal sepsis. Patient cells showed an absence of subagarose migratory response to activation serum (C5) but intact FMLP response. Superoxide production to FMLP was 5.3 +/- 0.1 nmole FcC compared to 11.2 +/- 0.1 nmole for laboratory normals. Addition of cytochalasin B reversed this. Scatchard analyses of patients' cells showed affinity of 55.5 +/- 0.6 as compared to 36.5 +/- 1.8 for controls. The number of receptors of cell was approximately 55,000 for both controls and patients. Internalization studies revealed significant increase in tritiated-FMLP uptake at room temperature. These results support the hypothesis that superoxide suppression is due to enhanced clearance of stimulant by enhanced internalization of the receptor-ligand complex.     

Primary inflammatory reaction in synovial fluid and tissue in rabbit immobilization osteoarthritis

The kinetics and composition of the primary cellular inflammatory process were studied in the synovial fluid (SF) and synovial tissue (ST) compartments of a rabbit knee immobilization osteoarthritis model. Immobilization induced rapid migration of neutrophils (59% +/- 26% of all cells) into SF in three days, which was accompanied by nonspecific esterase-positive monocytes (71% +/- 8% of all mononuclear cells). This finding suggests that non-specific inflammation mediated by phagocytic leukocytes predominates the cellular response in the SF compartment. In contrast, morphometric analysis of ST proper showed an inflammatory mononuclear cell response, the intensity of which diminished over time during the study period from Day 3 (416 +/- 59 cells per 0.049 mm2 ST tissue) through Day 10 (305 +/- 32 cells) to Day 35 (174 +/- 36 cells). A dotlike T-pattern alpha-naphthyl acetate esterase (ANAE) was found in the T-cell-dependent areas of secondary lymphatic tissue in the spleen, enabling immunocytologic ANAE marker studies. The ST response in situ was predominated by tissue macrophage, though infiltrates rich in T lymphocytes were present in the immediate sublining stroma. There was a significant correlation between the intensity of the SF cell response (total recovery) and the percentage of neutrophils, but there was no correlation between the intensity of the ST response and the proportion of T lymphocytes. These T-cell accumulations together with the local proliferation of fibroblastlike lining cells and stromal fibroblasts suggest that the primary inflammatory cell response is not caused by either wear and tear or mechanically by cartilage fragments.     

Left Heart Pump-Assisted Myocardial Revascularization Favorably Affects Neutrophil Apoptosis

Objectives  

Granulocyte apoptosis is a key control process in the clearance of neutrophils from inflammatory sites, and its rate is modulated by a number of inflammatory mediators. In this study, we investigated whether the use of left ventricular-assisted technique (LVA) in beating heart myocardial revascularization would exert less impact on neutrophil apoptosis compared with conventional cardiopulmonary bypass (CPB).     

Circulating neutrophils exhibit enhanced apoptosis associated with mitochondrial dysfunctions after surgery under general anaesthesia

BACKGROUND: Evidence suggests that apoptosis plays a main role in the postoperative changes detected in the polymorphonuclear neutrophil (PMN) population. Furthermore, recent studies have demonstrated that mitochondrial alterations constitute critical events of the apoptotic cascade. In this study we investigated whether apoptosis among neutrophils taken from patients undergoing surgical trauma could be associated with perturbation of mitochondrial transmembrane potential (deltapsim) and/or exaggerated production of mitochondrial reactive oxygen species (ROS). METHODS: Twenty-seven patients undergoing elective surgery under general anaesthesia were enrolled in the study. Peripheral blood samples were drawn one day before the operation and at 12 and 24 h after surgery. Apoptosis rate was assessed by staining neutrophils with 7-amino-actinomycin D (7-AAD) and by analysis by a FACScan flow cytometer. In order to evaluate deltapsim, cells were exposed to 3,3-dihexyloxacarbocyanine iodide [DiOC6(3)]; intracellular ROS was measured by means of hydroethidine (HE) and 2,7-diclorofluorescein diacetate (DCFH-DA), followed by analysis on a cytofluorometer. RESULTS: At 12 h following surgery we observed a significantly (P<0.05) increased frequency of apoptotic PMNs compared to that preoperatively (30.79+/-3.68% vs 7.40+/-0.69%). At this same time-point, the rate of neutrophils stained with HE, DCFH-DA and [DiOC6(3)] were significantly (P<0.05) higher compared to baseline (51.05+/-5.44%, 50.58+/-5.84% and 55.31+/-4.33% vs 20.17+/-2.38%, 19.59+/-2.03 and 25.43+/-2.71% respectively). Overall measurements returned to the preoperative values 24 h after surgery. CONCLUSION: These data suggest that surgery under general anaesthesia triggers in the immediate postoperative period pathways of PMN accelerated apoptosis associated with significant alterations in mitochondrial function.     

烟雾吸入性损伤对大鼠肺泡巨噬细胞吞噬功能及炎细胞凋亡的影响

目的 探讨烟雾吸人性损伤对大鼠肺泡巨噬细胞吞噬功能及炎细胞凋亡的影响。方法 选用54只Wistar大鼠，取其中6只作为正常对照组，其余48只制成烟雾吸人伤模型，作为吸人伤组。吸人伤组分别于致伤后2、6、12、24h及2、3、4、5d行支气管肺泡灌洗，获取肺泡巨噬细胞。观察(1)肺泡巨噬细胞体外对鸡红细胞吞噬功能的动态变化；(2)利用髓过氧化物酶(MPO)染色法观察肺泡巨噬细胞的染色阳性率；(3)利用流式细胞仪观察支气管肺泡灌洗液中炎细胞凋亡的动态变化。结果 (1)烟雾吸人伤早期(2～6h)肺泡巨噬细胞吞噬鸡红细胞功能受损，吞噬率下降，12h后逐渐恢复正常。(2)肺泡巨噬细胞MPO染色阳性率在吸人伤后逐渐升高，24h达峰值，2～5d逐渐下降。(3)支气管肺泡灌洗液中细胞凋亡率在3．02％～12．95％之间，以伤后24h凋亡率最高。结论 气管肺泡灌洗液中炎细胞凋亡增加，中性粒细胞凋亡及肺泡巨噬细胞吞噬凋亡细胞参与了烟雾吸人伤大鼠恢复期肺内炎症消散的过程。     

Modulation of neutrophil apoptosis in plasma of patients after orthognathic surgery

BACKGROUND: Human neutrophils undergo rapid apoptosis during in vitro culture. The aim of this study was to investigate the role of interleukin-8 (IL-8) on neutrophil apoptosis in surgery-induced inflammation. MATERIALS AND METHODS: Blood samples were drawn from 21 patients with mandibular prognathism 2 days before, and 1 and 5 days after orthognathic surgery. The IL-8 levels in the separated plasma were measured using an ELISA kit. The expression of two receptors for IL-8, CXCR1, and CXCR2, and their role in neutrophil apoptosis was evaluated using a flow cytometer. RESULTS: The IL-8 levels in the plasma were correlated with acute inflammatory markers, such as peripheral blood neutrophil counts and C-reactive protein levels. Both IL-8 receptors were markedly raised in patient-derived neutrophils 1 day post-operatively. Recombinant IL-8 (0-100 ng/ml) suppressed apoptosis in fresh-isolated neutrophils from healthy donors dose-dependently. Neutrophil apoptosis 1 day post-operatively was slightly accelerated in the presence of fetal bovine serum compared to the value 2 days pre-operatively and 5 days post-operatively. In contrast, in the presence of autogenous plasma, neutrophil apoptosis was significantly suppressed 1 day post-operatively compared to the value 2 days pre-operatively and 5 days post-operatively. Moreover, the anti-apoptotic effect of plasma on neutrophil apoptosis was partially decreased by the addition of anti-IL-8 neutralizing antibody. CONCLUSIONS: These results suggest that circulating neutrophils are susceptible to augmentation by IL-8 through the reinforcement of IL-8 receptors in acute inflammatory conditions. Furthermore, IL-8 may, in part, contribute to the regulation of neutrophil survival during the inflammatory response.     

Role of alveolar macrophages in asbestosis: modulation of neutrophil migration to the lung after acute asbestos exposure.

After intratracheal injection of short chrysotile asbestos fibres in guinea-pigs an intense neutrophil alveolitis was observed within three days. Evaluation by bronchoalveolar lavage of the inflammatory and immune effector cells producing the alveolitis by three days showed an increased proportion of polymorphonuclear leucocytes, which comprised 21% +/- 3% of the total leucocytes compared with 9% +/- 2% for the controls (p less than 0.05), persisting for at least six weeks (after which time the polymorphonuclear leucocytes comprised 28% +/- 2% compared with 7% +/- 1% for the controls: p less than 0.05). One mechanism by which asbestos fibres may cause polymorphonuclear leucocytes to be attracted to the alveolar structures is by induced release of neutrophil chemotactic factor by alveolar macrophages. When exposed in vitro to short or intermediate chrysotile fibres or amosite or crocidolite fibres guinea-pig alveolar macrophages released appreciable amounts of neutrophil chemotactic factor. The release of this chemotactic factor was augmented when the asbestos fibres had been previously exposed to normal serum. The chemotactic factor was lipid soluble, and was similar to the neutrophil chemotactic factor spontaneously released by alveolar macrophages recovered from guinea-pigs exposed in vivo to short chrysotile fibres. These observations suggest that alveolar macrophages may play an important part in the early stages of asbestosis by modulating the migration of neutrophils to the lung.     

Type 2 diabetic patients with nephropathy show structural-functional relationships that are similar to type 1 disease

Glomerular structural-functional relationships were investigated in 21 type 2 diabetic patients with proteinuria. Structural parameters were quantified using both light and electron microscopy and standard stereologic techniques. Data were also available on 14 nondiabetic subjects. Mesangial and matrix volume fractions and glomerular basement membrane (GBM) width were increased in type 2 patients when compared with nondiabetic subjects (mean +/- SD: 0.45 +/- 0.13 versus 0.18 +/- 0.03, P: < 0.001; 0.28 +/- 0.09 versus 0.10 +/- 0.02, P: < 0.001; and 665 +/- 138 versus 361 +/- 51 nm, P: < 0. 001, respectively). An increase in mesangial volume fraction was associated with high levels of proteinuria and low creatinine clearance (r = 0.64, P: = 0.002; r = -0.58, P: = 0.006, respectively). GBM width and mesangial foot process width (FPW(mes)) also correlated with proteinuria (r = 0.58, P: = 0.006; r = 0.60, P: = 0.004, respectively). Volume fraction of interstitium correlated with creatinine clearance (r = -0.58, P: = 0.006). Patients had previously been defined by light microscopy as having either diffuse or nodular glomerulosclerosis; those with nodules had larger mesangial and matrix volume fractions and more proteinuria than those classified as diffuse (mean +/- SD: 0.51 +/- 0.12 versus 0.36 +/- 0.08, P: = 0.007; 0.32 +/- 0.08 versus 0.21 +/- 0.05, P: = 0. 003; median, range: 4.3, 1.1 to 9.6 versus 1.1, 0.9 to 12.7 g/24 h, P: = 0.027). Creatinine clearance did not differ significantly between the groups. Type 2 diabetic patients with proteinuria have established glomerulopathy, which is more advanced in those with nodular glomerulosclerosis. Creatinine clearance correlated with both mesangial and interstitial expansion, whereas proteinuria correlated only with glomerular pathology. These results suggest that type 2 patients with advanced nephropathy have structural-functional relationships similar to type 1, consistent with a common pathogenesis, and strongly support an important role of the tubulointerstitium in the role of renal impairment.     

Neutrophil priming and apoptosis in anti-neutrophil cytoplasmic autoantibody-associated vasculitis

BACKGROUND: Interactions between anti-neutrophil cytoplasmic autoantibody (ANCA) and primed neutrophils (PMNs) may be central to the pathogenesis of primary small vessel vasculitis. PMNs from patients are primed, expressing proteinase 3 (PR3) on the cell surface, which permits interaction with ANCA. In vitro ANCA activates primed PMN to degranulate and generate a respiratory burst. Resultant reactive oxygen species are important in triggering apoptosis, but the fate of PMN in ANCA-associated vasculitis is unknown. Failure to remove apoptotic PMN in a nonphlogistic manner may sustain the inflammatory response. METHODS: PMNs from patients or controls were isolated, and the basal production of superoxide was measured by the superoxide dismutase-inhibitable reduction of ferricytochrome C. ANCA antigen expression on apoptotic PMN was assessed at 0, 12, and 18 hours by flow cytometry using dual staining with FITC-conjugated annexin V and PE-conjugated anti-murine IgG against monoclonal ANCA. Apoptosis was also assessed by morphology. In further studies, apoptotic PMNs were opsonized with monoclonal anti-myeloperoxidase (MPO) or anti-proteinase-3 (PR3) or irrelevant isotype-matched IgG (N IgG) and phagocytosis by macrophages was measured using interaction assays. Cytokines interleukin-8 (IL-8) and interleukin-1 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Proteinase-3 expression (active 63.04 +/- 5.6% of total number of cells, remission 51.47 +/- 7.9% of total number of cells, control 17.7 +/- 4.7% of total number of cells, P < 0.05) and basal superoxide production (active 6.9 +/- 0.8 nmol/L x 10(6) cells, remission 5.15 +/- 0.4 nmol/L/10(6) cells, control 3.63 +/- 0.3 nmol/L/10(6) cells, P < 0.001) were significantly greater with freshly isolated PMN from patients than controls. PR3 expression and superoxide generation were positively correlated. PMN from patients with active disease became apoptotic at a greater rate than those of controls (at 18 hours, patients 72.3 +/- 3.9% apoptosis, controls 53.2 +/- 2.7% apoptosis, P < 0.05). PR3 and MPO expression were significantly greater on PMN isolated from patients at 12 and 18 hours. Opsonization of apoptotic PMN with ANCA significantly enhanced recognition and phagocytosis by scavenger macrophages (anti-MPO 88.95 +/- 6.27, anti-PR3 93.98 +/- 4.90, N IgG 44.89 +/- 3.44, P < 0.01) with increased secretion of IL-1 (anti-PR3 34.73 +/- 6.8 pg/mL, anti-MPO 42.01 +/- 12.3 pg/mL, N IgG 8.04 +/- 6.3 pg/mL, P < 0.05) and IL-8 (anti-PR3 8.97 +/- 0.93 ng/mL, anti-MPO 8.45 +/- 1.46 ng/mL, N IgG 0.96 +/- 0.15 ng/mL, P < 0.01). CONCLUSION: In vivo circulating PMNs are primed as assessed by PR3 expression and basal superoxide production, thereby enhancing their inflammatory potential. These PMNs undergo apoptosis more readily, at which times they express PR3 and MPO on their surface. These antigens may then provide targets for ANCA. Opsonization of apoptotic PMN will enhance clearance by macrophages but will also trigger the release of pro-inflammatory cytokines that may contribute to chronic inflammation.