Modulation of postsurgical macrophage function by postsurgical polymorphonuclear leukocytes]

Rapid and transient influx of polymorphonuclear leukocytes (PMN) is observed prior to accumulation of macrophages after surgical trauma. Rabbits underwent intestinal reanastomosis and at various times peritoneal exudate cells were collected and separated using a Percoll gradient. Postsurgical macrophages were incubated with PMN spent media obtained from various postsurgical periods. Macrophage release of O2- had already increased at 2 hr after surgery, reached peak levels at 6 hr and decreased by 24 hr. PMN spent media from 6 hr postsurgical cells functioned as a suppressor, whereas 12 or 24 hr PMN spent media increased the O2- release from the macrophages harvested at 6 and 12 hr after surgery. Plasminogen activator (PA) activity in the macrophage spent medium was elevated at 24 hr after surgery by exposure to PMN spent media, however no effects were observed on macrophages harvested within 12 hr after surgery. PA inhibitory activity was reduced at 2 hr after surgery, and gradually increased, but no effects of PMN spent media on the PA inhibitory activity was observed. Thus, soluble factors secreted into the medium by PMN may modulate macrophage metabolism in stages as the macrophages differentiate and promote wound repair.     

Modulation of postsurgical macrophage function by early postsurgical polymorphonuclear leukocytes.

Surgical trauma to the peritoneum, in the absence of infection, elicits a rapid and transient influx of polymorphonuclear leukocytes (PMNs) into the peritoneal cavity prior to the accumulation of macrophages. The aim of this study was to characterize the effects of these PMNs on macrophage function in the early postsurgical period. Rabbits underwent intestinal reanastomosis and peritoneal exudate cells were collected at various times after surgery. Macrophage-enriched preparations were incubated with spent media from cultures of PMNs obtained at the corresponding times after surgery. Superoxide anion (O2-) release by macrophages in response to phorbol myristate acetate was determined by cytochrome c reduction. Fibrinolytic and protease inhibitory activities in macrophage-spent media were also evaluated. The release of O2- had already increased at 2 hr, reached peak levels at 6 hr, and decreased by 24 hr after surgery. Spent media from PMNs harvested 6 hr after surgery suppressed, whereas spent media from postsurgical 12- or 24-hr PMNs increased O2- release from macrophages harvested at 6 and 12 hr after surgery. PMN-spent media had no effect on the secretion of plasminogen activator (PA) from macrophages harvested within 12 hr after surgery. In contrast, PA activity in the spent media from macrophages harvested 24 hr after surgery was elevated after exposure to PMN-spent media. PA inhibitory activity was reduced in macrophage-spent media at 2 hr after surgery and increased by 24 hr, while PMN-spent media had no effect on the level of PA inhibitory activity. Thus, soluble factors secreted into the culture medium by PMNs modulate macrophage function as soon as 6-12 hr after surgery.     

Modulation of peritoneal re-epithelialization by postsurgical macrophages.

The studies reviewed here show that postsurgical macrophages are capable of modulating the proliferation of TRC. That is, macrophages either suppress or enhance the proliferation of TRC depending on the culture time and the medium used as a comparison, i.e., culture medium with only serum or spent medium from cultures of resident peritoneal macrophages. Postsurgical macrophages also modulate the morphology of (spindly or rounded appearance) and the secretion of extracellular matrices by TRC. The responsivity of TRC to control by postsurgical macrophage-spent media or growth factors changes as a function of postsurgical and/or culture time. In addition, cells harvested from the site of peritoneal trauma (TRC) did not respond to growth factors in a fashion entirely the same as fibroblasts. This indicates that cells harvested from the site of peritoneal injury are unique. Lastly, after removal of a suppressive factor from postsurgical macrophage-spent media by dialysis, the factors secreted by postsurgical macrophages are more potent in enhancing TRC proliferation than growth factors individually.     

NO在小鼠烧伤后腹腔巨噬细胞功能变化中的作用

目的探讨ＮＯ在烧伤后小鼠巨噬细胞（ＭΦ）功能变化中的作用。方法通过ＮＭＭＡ等的应用,观察体外培养烧伤后腹腔ＭΦ（ＰＭΦ）产生ＮＯ、ＰＧＥ２和ＴＮＦα的变化,以及对烧伤后ＰＭΦ抑制脾淋巴细胞（ＳＬ）增殖的影响。结果烧伤早期ＭΦ就能产生大量ＮＯ,ＮＯ能促进烧伤后ＰＭΦ产生ＰＧＥ２及ＴＮＦα,且烧伤后２４小时（２４ＰＢＨ）ＰＭΦ可通过产生的ＮＯ显著抑制ＣｏｎＡ刺激的ＳＬ增殖。结论ＮＯ在烧伤后ＭΦ功能变化乃至机体的免疫功能紊乱中起着重要作用。     

肺泡巨噬细胞活化在急性坏死性胰腺炎大鼠肺损伤中的作用

目的　探讨肺泡巨噬细胞活化在急性坏死性胰腺炎 (ANP)肺损伤中的作用。　方法30只成年SD大鼠随机分为正常对照组、ANP后 1、3、6、12h组 ,每组 6只。逆行性胰胆管注射 3%牛磺酸钠建立ANP大鼠模型 ,正常对照组大鼠自胆胰管内逆行注入生理盐水。经支气管肺泡灌洗获取肺泡巨噬细胞 ,检测支气管肺泡灌洗液中蛋白含量、肺组织髓过氧化物酶 (MPO)水平、肺泡巨噬细胞分泌肿瘤坏死因子α(TNFα)及一氧化氮 (NO)水平。以反转录聚合酶链反应 (RT PCR)法测定肺泡巨噬细胞TNFαmRNA、诱导型一氧化氮合酶 (iNOS)mRNA表达情况。行肺、胰腺组织病理学检查并评分。结果　ANP大鼠肺损伤随着病情进展而逐渐加重 ;肺组织MPO及支气管肺泡灌洗液中蛋白含量逐渐升高 ,12h达最高值 ,分别为 (10 78± 0 5 8)U/g和 (2 0 11 0± 10 5 5 ) μg/ml;肺泡巨噬细胞分泌TNFα、NO水平逐渐升高 ,至 6h达到高峰 ,分别为 (16 2 4 2± 149 2 )pg/ml和 (88 8± 6 5 ) μmol/L ,12h又回落。ANP发生后 ,肺泡巨噬细胞TNFαmRNA、iNOSmRNA的表达情况与TNFα、NO的变化趋势相似。ANP大鼠各组指标与正常对照组相比差异均有显著性意义 (P <0 0 5 )。组织学评分结果表明 ,随着胰腺损伤的加重肺损伤也逐渐加重。肺泡巨噬细胞TNFαmRNA、iNOSmRNA     

Toll-like receptor 4 plays a role in macrophage phagocytosis during peritoneal sepsis

Background

Peritoneal sepsis is a significant cause of mortality in infants with necrotizing enterocolitis, caused in part by impaired bacterial clearance. Recent studies have identified toll-like receptor-4 (TLR4) as a receptor for endotoxin (lipopolysaccharide [LPS]). We hypothesized that TLR4 regulates bacterial clearance from the peritoneal cavity and sought to investigate whether macrophage phagocytosis was involved.

Methods

Peritoneal sepsis was induced in mice expressing either functional TLR4 (TLR4-wild-type [WT]) or mutant TLR4 by intraperitoneal injection of either live Escherichia coli or LPS. Phagocytosis was assessed by measuring the uptake of opsonized red cells. To assess bacterial clearance, we irrigated peritoneal cavities of injected animals with saline and plated it on gram-negative selective media.

Results

LPS significantly increased the rate of phagocytosis by peritoneal macrophages from TLR4-WT mice, but not in those from TLR4-mutant mice, suggesting a role for TLR4 in phagocytosis. LPS also increased the rates of phagocytosis in cultured macrophages expressing TLR4, confirming these findings. The yield of gram-negative bacteria obtained from the peritoneal cavities of septic TLR4-WT mice was greater than that from TLR4 mutants, consistent with TLR4-dependent alterations in their septic course.

Conclusions

We conclude that TLR4 plays a critical role in the response to intraperitoneal E. coli through effects on phagocytosis by macrophages, suggesting the possibility of using TLR4 as a therapeutic target in diseases of peritoneal sepsis.     

Mitogenic and protein synthetic activity of tissue repair cells: control by the postsurgical macrophage

It is well known that fibroblasts are a main source of extracellular matrix synthesis necessary for tissue repair. In addition, macrophages secrete products that are known to modulate synthesis of extracellular matrix. Accordingly, we studied the incorporation of [3H]thymidine, [3H]proline, and [35S]sulfate into macromolecules produced by fibroblasts recovered from the site of peritoneal tissue repair cultured with and without spent media from postsurgical peritoneal macrophages. Rabbits underwent resection and reanastomosis of their small intestines. Peritoneal exudative cells (PEC) were then collected on postsurgical day 5 and day 10 as well as from nonsurgical controls, separated by discontinuous Percoll gradient centrifugation, and cultured for 48 h. A second group of rabbits underwent peritoneal wall abrasion from which fibroblast tissue repair cells (TRC) were collected from the site of injury at postsurgical day 7 and maintained in culture for varying times. Incorporation of radiolabeled precursors into DNA, collagen, and sulfated proteoglycans was determined. Incorporation of [3H]thymidine and [3H]proline into untreated TRC gradually decreased with culture duration. Conversely, [35S]sulfate incorporation gradually increased during prolonged culture. Macrophage spent media increased the levels of [3H]thymidine incorporation by the TRC. [3H]Proline and [35S]sulfate incorporation into TRC were also stimulated by macrophage spent media. However, this stimulation may be due to the enhanced proliferation of TRC by macrophage spent media. In conclusion, tissue repair fibroblasts are activated for postsurgical repair at the site of injury by many factors including secretory products from postsurgical macrophages.     

A case report of double roentgenographically occult lung cancer revealed by postsurgical pathological study]

A 66-year-old man admitted to our hospital as a roentgenographically occult lung cancer (ROLC) detected by sputum cytology of class IV. A differential brushing of all branches of the bronchi was performed and squamous cell carcinoma was detected only from the right B8 segmental bronchus. Right lower lobectomy was performed and the microscopic findings of surgical specimen revealed the squamous cell carcinomas were seen at not only B8 bronchus but also B7 bronchus. The frequency of multicentricity of ROLC is reported to be high, and a differential bronchial brushing of all bronchi is a very powerful method to diagnose synchronous multiple lung cancer. However, we failed to detect a cancer lesion of B7 segmental bronchus in this case. Since, the outcomes of surgical treatments for either synchronous or metachronous multiple primary lung cancer are satisfactory, limited surgical treatments might be appropriate as an initial treatment for a ROLC.     

The role and mechanism of macrophage activation induced by exosomes from high glucose-treated renal tubular epithelial cells

Objective To investigate the role and mechanism of macrophage activation induced by exosomes from high glucose-treated renal tubular epithelial cells. Methods (1) The supernatant of renal tubular epithelial cells which were cultured in normal glucose control group (5.5 mmol/L D-glucose) or high glucose group (30.0 mmol/L D-glucose) for 48 h were collected and ultracentrifuged to harvest exosomes. Exosomes were identified by transmission electron microscope and Western blotting. (2) Exosomes were labeled with the green lipophilic fluorescent dye PKH67 and cultured with THP-1 macrophage to investigate whether HK2-derived exosomes could be internalized by THP-1 macrophage. Observing the morphology microscopically and detecting the chemotaxis function of THP-1 macrophages in Transwell chamber after co-cultured with exosomes. The expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1) in cells and supernatants were separately detected by quantitative Real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA), and the expression of p-c-Jun NH2-terminal kinase (p-JNK), mitogen-activated protein kinase p-p38 (p-p38MAPK) and nuclear factor κB p65 (NF-κB p65) in THP-1 macrophages were detected by Western blot. Results (1) Vesicles that harvested by ultracentrifugation ranged in size from 30 nm to 100 nm and expressed exosomal marker CD63, TSG101 but absence of calnexin which is a marker of endoplasmic reticulum, suggesting that the exosomes were not contaminated with cells. (2) Results from laser scanning confocal microscope showed that each group of exosomes can be internalized by THP-1 macrophages. Compared with normal glucose exosomes group, high glucose exosomes had increased the expression of iNOS, TNF-α, IL-1β and MCP-1 in THP-1 macrophages (all P＜0.01), moreover, p-JNK, p-p38 MAPK and NF-κB p65 proteins level also increased significantly (all P＜0.01). Conclusions Exosomes from high glucose-treated HK2 cells can induce THP-1 macrophage activation and functional changes through MAPK/NF-κB pathway.     

腹膜透析微炎症状态诱导腹膜损伤的中医治疗策略

腹膜透析(PD)患者普遍存在不同程度的微炎症状态,PD微炎症状态参与并介导了腹膜损伤,促进腹膜纤维化的发生发展,严重影响PD患者生活质量及生存率。目前缺乏特异性防治PD微炎症状态的临床药物。传统医学认为,脾气亏虚、毒损腹络是PD微炎症状态诱导腹膜损伤的主要病机,健脾益气法可以防治PD微炎症状态诱导的腹膜损伤,保护腹膜功能。在中医理论指导下,结合现代医学对PD微炎症状态诱导的腹膜损伤发生机制的认识,形成中医药防治PD微炎症状态相关性腹膜损伤的理法方药体系,将为中医药防治PD微炎症状态提供新方法。     

The role of NO in macrophage dysfunction at early stage after burn injury

AIM: To explore the role of nitric oxide (NO) in macrophage dysfunction at early stage after burn injury. METHOD: Peritoneal macrophages were isolated and cultured from early stage burnt mice. NO production and inducible NO synthase (iNOS) expression in the macrophages were checked by the Greiss method and real-time PCR (TaqMan), respectively. l-Arginine, the substrate of NO producing, or N-monomethyl-l-arginine (l-NMMA), a competing blocker of NOS was administered to the culture, the changes of NO, TNF-alpha and PGE2 productions were measured, additionally the changes of the iNOS, TNF-alpha and COX-2 expression were assayed by real-time PCR. After that, the effects of l-arginine and l-NMMA were determined on burnt macrophage influencing the proliferation of normal splenic lymphocytes. RESULT: A large amount of NO was produced by macrophages from post burn hour 6 (6PBH) with a high level of iNOS expression. l-Arginine could increase NO production in a dosage-dependent manner, while l-NMMA attenuated NO production, but neither could affect iNOS expression. Moreover, l-arginine enhanced productions of both the latter produced TNF-alpha and PGE2 from burnt macrophages, and the expressions of TNF-alpha and COX-2 were improved significantly, while l-NMMA did reverse ways. It was found that macrophages from post burn hour 24 mice could inhibit Con A-stimulated normal splenic lymphocytes dramatically, l-NMMA could decrease this function significantly, but l-arginine could not influence the suppression. CONCLUSION: Our experiment indicated NO derived from burnt macrophage played a vital role in macrophage producing excessive TNF-alpha and PGE2, and suppressing lymphocyte function at early stage after burn injury.     

Proximal tubule cells stimulated by lipopolysaccharide inhibit macrophage activation

BACKGROUND: Tubule cells can produce a variety of cytokines, extracellular matrix (ECM) components, and adhesion molecules in vitro and in vivo. It is generally assumed that stimulated tubule cells are proinflammatory and at least partially responsible for interstitial inflammation. However, the overall effect of tubular cells on interstitial cells is unknown. In this study, pro- and anti-inflammatory cytokine production and net effects on macrophages of tubule cells activated by lipopolysaccharide (LPS) were examined. METHODS: Tubule cells stimulated with LPS expressed tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-12, monocyte chemoattractant protein-1 (MCP-1), IL-10, and transforming growth factor-beta (TGF-beta). Conditioned media were collected from confluent monolayers of rat tubule cells stimulated, or not, by LPS for 4 and 18 hours, respectively. Macrophages were cultured with conditioned media and/or LPS (0.5 microg/mL) for 18 hours. RESULTS: TNF-alpha and IL-lbeta mRNA of macrophages stimulated by LPS increased more than fivefold when cultured with control conditioned media from unstimulated tubule cells. Surprisingly, TNF-alpha and IL-lbeta levels of macrophages stimulated by LPS were not increased when cultured with conditioned media from activated tubule cells. Neutralizing antibodies to IL-10 and TGF-beta were used to define the inhibitory component(s) in conditioned medium. Anti-IL-10, but not anti-TGF-beta, abolished partially the inhibitory effects of conditioned media on macrophages. CONCLUSION: Tubule cells produce both pro- and anti-inflammatory cytokines and the net effect, partially explained by IL-10, of tubule cells activated with LPS is to inhibit activity of macrophages. Thus, the net effect of activated tubule cells on interstitial pathology may in certain circumstances, be anti- rather than pro-inflammatory.     

Development of peritoneal adhesions in macrophage depleted mice

BACKGROUND: We present a new mouse model for the study of peritoneal adhesions using macrophage Fas-induced apoptosis (Mafia) transgenic mice expressing a Fas-FKBP construct under control of the murine c-fms promoter. Mafia mice allow systemic macrophage depletion by dimerization of Fas with a synthetic dimerizer, AP20187. Results demonstrate that macrophage depletion in Mafia mice induces peritoneal adhesion formation when the peritoneal cavity is also exposed to an irritant. The Mafia mouse model presents a reproducible, non-surgical approach for research in adhesion formation and prevention. MATERIALS AND METHODS: Mafia mice were treated with AP20187 using an intravenous (i.v.) or intraperitoneal (i.p.) injection. Control groups included mock-treated Mafia mice and both AP20187 and mock-treated wild type mice. Seven days after treatment, mice were observed for the presence of adhesions. RESULTS: After i.p. injection with AP20187, 76% of Mafia mice developed adhesions whereas none of the mock-treated Mafia or wild-type mice developed adhesions, and only one AP20187-treated wild-type mouse (5.8%) developed a mild adhesion. Mafia mice treated with AP20187 i.v. exhibited macrophage depletion not significantly different than i.p. treated mice, but did not develop adhesions. In contrast, Mafia mice treated with AP20187 i.v. developed adhesions when diluent was also injected into the peritoneal cavity, whereas i.p diluent alone had no effect. CONCLUSION: Macrophage depletion, combined with a peritoneal irritant, results in peritoneal adhesion formation in transgenic Mafia mice. Macrophages appear to play a protective role in the development and/or repair of peritoneal adhesions.     

The role of peritoneal lavage in treatment of penetrating abdominal injuries]

Preventing negative laparotomies is one of the most challenging problems in the management of penetrating abdominal injuries. The term "selective laparotomy" has been therefore introduced and has found an ever increasing acceptance. The peritoneal lavage is a useful tool in patient selection for laparotomy but the main problem is where to set the boundary between a positive and a negative peritoneal lavage. The manipulation of this boundary leads to significant changes in the sensitivity and specificity of the peritoneal lavage. Here we are presenting 162 consecutive cases of penetrating abdominal trauma and discussing our methods of evaluation and management.     

不同免疫调节剂对烫伤大鼠巨噬细胞功能的影响

目的 探讨不同免疫调节剂对烫伤大鼠腹腔巨噬细胞（Mφ）功能的影响。方法 应用McAb APAAP桥联酶标法、琼脂溶菌板法和MTT比色法等技术，分别测定了各组烫伤大鼠不同时相腹腔Mφ各种功能的变化。结果 ①烫伤后大鼠腹腔Mφ表面Ia抗原表达率降低，抗原提呈能力减弱，对念珠菌的噬菌率下降，溶菌酶活力减低，TNF分泌增高，且伤后第10天比第5天更明显。以上结果与正常组比较，差异有非常显著意义（P＜0．01）。②经不同免疫调节剂治疗后，各组烫伤大鼠腹腔Mφ各种功能明显改善，各项指标与未用药的对照组,差异有非常显著意义（P＜0．01）。结论 烫伤后早期腹腔注射特异性免疫核糖核酸（iRNA)可明显改善伤鼠的免疫功能。     

Cellular distribution of 5-lipoxygenase and leukotriene receptors in postsurgical peritoneal wound repair

Products of the eicosanoid pathways, namely prostaglandins and leukotrienes, are known to play a key role in inflammatory and immune responses, as well as other wound cellular activities of wounded tissue. The objective of this study was to determine whether surgically induced intraperitoneal and incisional wounds in the rat express 5-lipoxygenase and contain binding sites for leukotrienes and whether their levels change during the course of healing. With the use of a specific monoclonal antibody generated against 5-lipoxygenase, the enzyme was immunohistochemically localized in various wound cells during postsurgical days 2 to 35. The inflammatory cells within the wound were the major cell types containing 5-lipoxygenase immunoreactive protein, followed by fibroblasts in the incisional and peritoneal fibrous adhesions, striated muscle, and the vasculature. The greatest level of immunostaining was observed during the first 2 weeks after surgery, which decreased to near unwounded levels by day 35. Light microscope autoradiographic binding studies using (3)H-leukotrienes indicated that the peritoneal/incisional wounds and unwounded tissues contain specific (3)H-leukotriene C(4) and (3)H-leukotriene D(4) but not (3)H-leukotriene B(4) binding sites. Quantitative grain analysis (net grain density/100 microm(2)), representing specific (3)H-leukotriene C(4) and (3)H-leukotriene D(4) binding sites calculated for different cell types in the wound and unwounded regions showed that (3)H-leukotriene C(4) binding was highest over the striated muscle proximal to the injury and incisional and peritoneal granulation tissue fibroblasts. The net grain density over these cells increased by 3-, 2.5-, and 2-fold by day 14, respectively, and declined to the control values by day 21 after injury (p < 0.05). The pattern of (3)H-leukotriene D(4) binding was similar to that observed for (3)H-leukotriene C(4), but with a lower density. The grain density for (3)H-leukotriene C(4) and (3)H-leukotriene D(4) in arteriolar endothelial and smooth muscle cells remained unchanged. These data suggest that the products of the lipoxygenase pathway through the presence of their specific receptors may play an important role in peritoneal wound repair and adhesion formation.     

Apparent successful mesothelial cell transplantation hampered by peritoneal activation

BACKGROUND: Mesothelial cell transplantation has been suggested to improve mesothelial repair after surgery, recurrent peritonitis and peritoneal dialysis. METHODS: In this study we evaluated mesothelial cell transplantation during the resolution phase of experimentally thioglycollate-induced peritonitis in rats. To this end 4 x 10(6) DiO-labeled autologous mesothelial cells were transplanted 1 week after peritonitis induction. Peritoneal inflammation and permeability characteristics were evaluated after another week. RESULTS: Mesothelial cell transplantation after peritonitis resulted in incorporation of these cells in the parietal mesothelial lining, leading to an acute transient submesothelial thickening which was not seen in transplanted animals without prior peritonitis induction. Long-term functioning of these repopulated mesothelial cells leaded to peritoneal activation as evidenced by a approximately twofold increase in peritoneal lymphocytes (P < 0.01) and omental mast cell counts (P < 0.05), accompanied by the induction of inflammation markers monocyte chemoattractant protein-1 (MCP-1) (P < 0.01) and hyaluronan (P < 0.01) in the transplanted peritonitis group, but not in rats with peritonitis without mesothelial cell transplantation or in control rats without mesothelial cell transplantation (all four parameters P < 0.01). In addition, trapping of transplanted mesothelial cells in the milky spots of omental tissue and lymphatic stomata of the diaphragm both in control and thioglycollate rats seems to increase microvascular permeability, reflected by apparent increased diffusion rates of small solutes and proteins. CONCLUSION: Altogether, our data underscore the importance of controlling peritoneal (patho)physiology and function in mesothelial transplantation protocols.