Immunohistochemical analysis of markers for different macrophage phenotypes and their use for a forensic wound age estimation

A total of 117 vital skin wounds (post infliction intervals between a few seconds and 7 months), 20 postmortem wounds and skin specimens with beginning or advanced signs of putrefaction were investigated. Different markers for macrophage maturation (27 E 10, RM 3/1, 25 F 9, G 16/1) were analyzed by immunohistochemistry. The early stage inflammation marker 27 E 10 stained macrophages, but also monocytes and neutrophilic granulocytes localized in blood vessels or bleeding induced postmortem and therefore provided no further information for a forensic wound age estimation in comparison to the routine histological detection of macrophages. The antigens recognized by the RM 3/1- (intermediate stage inflammation marker) and 25 F 9-antibodies (late stage inflammation marker) were expressed exclusively by histiocytes and inflammatory cells that had migrated from the blood vessels as part of the acute inflammatory response associated with an intravital reaction. The morphometrical analysis revealed positive results (defined as at least a two-fold increase in number in 2 or more microscope fields when compared to the maximum value of histiocytes found in uninjured skin) for the RM 3/1- or 25 F 9-antibody earliest in wounds aged 7 or 11 days, respectively. Similarly to the 25 F 9-antibody, the chronic stage inflammation marker (G 16/1) reacted with a macrophage subpopulation first detectable 12 days after wounding but showed positive results in a comparably reduced percentage of cases. On the other hand, this marker did not stain a relevant number of resident macrophages thus facilitating the evaluation of the specimens. The markers 27 E 10, RM 3/1 and 25 F 9 are also useful for the evaluation of slightly - even though the staining intensity was considerably reduced - but not advanced putrefied skin. Therefore, the immunohistochemical analysis of the corresponding antigens can possibly contribute to an age estimation of wounds with advanced post infliction intervals obtained from corpses with longer - but limited - postmortem intervals.

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Zusammenfassung Insgesamt wurden 117 vitale Hautwunden (Überlebenszeit wenige Sekunden bis 7 Monate), 20 postmortal gesetzte Verletzungen sowie Haut mit leichten bzw. fortgeschrittenen Fäulnisveränderungen untersucht und verschiedene Marker der Makrophagen-Differenzierung (27 E 10, RM 3/l, 25 F 9 und G 16/1) analysiert. Der early stage inflammation marker 27 E 10 färbte neben Makrophagen auch Monozyten und neutrophile Granulozyten, die innerhalb von Blutgefäßen bzw. in postmortal gesetzten Blutungen lokalisiert waren und liefert somit keine Informationen zum Wundalter, die über die Möglichkeiten des Routine-histologischen Nachweises von Makrophagen hinausgingen. Die von den Antikörpern RM 3/1 (intermediate stage inflammation marker) und 25 F 9 (late stage inflammation marker) erkannten Antigene wurden ausschließlich von Histiozyten und reaktiv eingewanderten Makrophagen exprimiert. Die morphometrische Analyse ergab positive Ergebnisse (definiert als ein mindestens zweifacher Anstieg der Zellzahl in zwei oder mehr Gesichtsfeldern verglichen mit der maximal feststellbaren Zahl an Histiozyten in unverletzter Haut) bei Verwendung der Antikörper RM 3/1 bzw. 25 F 9 frühestens 7 bzw. 11 Tage nach Wundsetzung. Ab 12 Tagen Wundalter reagierte der chronic stage inflammation marker G 16/1 erstmals positiv. Das Antigen ließ sich insgesamt allerdings in einem geringeren Prozentsatz der untersuchten Wunden darstellen. Vorteilhaft ist jedoch das Fehlen einer relevanten Expression durch Histiozyten, wodurch die Auswertung der Präparate erleichtert wird. Die entsprechenden Antigene lassen sich zudem in leicht - wenn auch in einer deutlich geringeren Färbeintensität -, aber nicht forgeschritten fäulnisveränderter Haut nachweisen, so daß deren immunhistochemische Darstellung gegebensfalls auch zur Beurteilung von länger überlebten Verletzungen an Leichen mit etwas fortgeschrittener Liegezeit herangezogen werden kann.

     

Immunohistochemical dynamics of leukotoxin (9.10-eooxv-12-octadecenoic acid) in lungs of rats

This paper investigates the immunohistochemical dynamics of leukotoxin (9,10-epoxy-12-octadecenoic acid, LTx) in the lungs of rats exposed to hyperoxia with or without paraquat. The rats were treated with 100% oxygen or ambient air for 24. 48, 72 and 96 h in the presence or absence of a low or high dose paraquat (1,1-di-methyl-4,4-bipyridinium, PQ) injection. Immunostaining for LTx demonstrated positive reactions in the neutrophils that showed a progressive increase in intensity of staining with time in all groups exposed to 100% oxygen and in the group with high dose PQ, but the positive findings were weak in the group injected with low dose PQ only. We found the positive immunostaining reaction not only in neutrophils but also in alveolar macrophages. This indicates that LTx is produced by alveolar macrophages as well as by neutrophils depending on the treatment period under hyperoxic conditions, suggesting that LTx is an important chemical mediator in pulmonary diseases.     

脾切除对小鼠腹腔巨噬细胞数量和功能的影响

目的：研究脾切除对小鼠腹腔巨噬细胞数量和功能活性的影响。方法：术后4周获取腹腔常居巨噬细胞和炎症巨噬细胞,检测其数量、吞噬功能和一氧化氨水平。结果：与正常对照组和假手术组比较,脾切除组小鼠的2种巨噬细胞不仅数量明显减少,而且它们的吞噬功能和一氧化氮水平呈明显下降。结论：提示脾切除造成的单核吞噬细胞继发性炎症反应的捐伤和巨噬细胞功能活性的降低,可能是脾切除后(凶险性)感染的重要因素。     

支气管哮喘患者肺泡巨噬细胞释放内皮素-1的研究

为研究支气管哮喘患者肺泡巨噬细胞（Ａｍ）所释放的内皮素１（ＥＴ１）在哮喘发病中的作用,对发作期哮喘患者２０例,对照组８例,经纤支镜行支气管肺泡灌洗术获得Ａｍ,调整Ａｍ至１×１０６／ｍｌ后分３孔体外培养（１ｍｌ／孔）;生理盐水对照孔、氟美松孔（１μｇ／ｍｌ）、氨茶碱孔（１５μｇ／ｍｌ）,培养６ｈ后取上清液,采用放免法测定ＥＴ１。显示哮喘组血浆ＥＴ１明显高于对照组,并于第１秒用力肺活量（ＦＥＶ１．０）呈显著负相关。哮喘组Ａｍ体外释放ＥＴ１与对照组无显著差异,与ＦＥＶ１．０无相关性。氟美松孔Ａｍ释放ＥＴ１明显低于生理盐水对照孔;氨茶碱孔与生理盐水孔或氟美松孔无差异。提示哮喘Ａｍ所释放ＥＴ１在哮喘发病中意义不大     

The role of VCAM-l/VLA-4 in the activation of allogenic T cells by murine macrophages

Vascular cell adhesion molecule 1 (VCAM-1) is a member of immunoglobulin superfamily. The principal ligand for VCAM-1 is integrin α4β/VLA-4 (very late antigen 4). It was reported that VCAM-1 was expressed on macrophages and dendritic cells, but little is known about its function on these professional antigen presenting cells (APC). The present study was performed to investigate the expression of VCAM-1 on macrophages and the role of VCAM-l/VLA-4 in the activation of allogenic T cells by murine macrophages. We analyzed VCAM-1 expression on peritoneal macrophages and macrophage cell line J774A.1 by fluorescence-activated cell sorting (FACS). Using neutralizing antibodies, we further analyzed the role of VCAM-l/VLA-4 interaction in macrophage and allogenic T cell mixed lymphocyte reaction (MLR). We found that VCAM-1 was constitutively expressed on macrophages and its expression level was upregulated by soluble tumor associated antigen (freeze-thaw lysates of FBL-3 leukemia cells) and TNF-a. In MLR assays, we observed that blocking VCAM-l/VLA-4 interaction with anti-VCAM-1 or anti-VLA-4 mAbs caused significant inhibition of the proliferative response and IL-2 production. These results suggest that VCAM-lon macrophages not only facilitates the cell-to-cell contact through adhesive interaction but also plays a role in the costimulation of T cells via its interaction with VLA-4 on the T cells. This work was supported by grants from the National Natural Science Foundation of China.No. (39730420). This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997).     

Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A(2).

The in vitro effects of myotoxin III (MT-III), an Asp-49 catalytically-active phospholipase A(2), and myotoxin II (MT-II), a catalytically-inactive Lys-49 variant, isolated from Bothrops asper snake venom, on phagocytosis and production of hydrogen peroxide (H(2)O(2)) by thioglycollate-elicited macrophages were investigated. MT-II and MT-III were cytotoxic to mouse peritoneal macrophages at concentrations higher than 25 microg/ml. At non-cytotoxic concentrations, MT-II stimulated Fcgamma, complement, mannose and beta-glucan receptors-mediated phagocytosis, whereas MT-III stimulated only the mannose and beta-glucan receptors-mediated phagocytosis. Moreover, both myotoxins induced the release of H(2)O(2) by thioglycollate-elicited macrophages, MT-III being the most potent stimulator. MT-II induced the release of H(2)O(2) only at a concentration of 3.2 microg/ml (130% increment) while MT-III induced this effect at all concentrations tested (0.5-2.5 microg/ml; average of 206% increment). It is concluded that, at non-cytotoxic concentrations, MT-II and MT-III activate defense mechanisms in macrophages up regulating phagocytosis, mainly via mannose and beta-glucan receptors, and the respiratory burst.     

Microcystin-LR promote intestinal secretion of water and electrolytes in rats.

We showed previously that exposure to microcystin-LR causes renal toxic effects in isolated perfused rat kidney, and that inflammatory mediators from supernatants of macrophages stimulated by microcystin-LR are involved in this process. The aim of this research was to examine water and electrolytes secretion in vivo, induced by microcystin-LR and supernatant of macrophages stimulated for this toxin (SUP.MphiS + MCLR), using perfused rat ileal segment and ligated intestinal loop models. We found microcystin-LR at 1 microg/ml (0.09 +/- 0.003* vs. control 0.07 +/- 0.001 g of secretion/2 cm of loop; P < 0.05*) and the SUP.MphiS + MCLR after 18 h postinoculation (0.10 +/- 0.003 vs. control 0.03 +/- 0.002 g/cm) caused intestinal secretion. In addition, microcystin-LR caused significant sodium secretion (-2.18 +/- 0.72* vs. control 2.18 +/- 0.50 microEq g(-1) min(-1)), potassium (-0.26 +/- 0.04* vs. control 0.32 +/- 0.03 microEq g(-1) min(-1)), chloride (MCLR = -3.29 +/- 1.93* vs. control 0.88 +/- 1.25 microEq g(-1) min(-1)) and water (-0.012 +/- 0.004* vs. control 0.002 +/- 0.002 ml g(-1) min(-1)). We also demonstrated SUP.MphiS + MCLR to induce intestinal secretion of electrolytes (sodium, potassium, chloride) and water. These findings suggested that microcystin-LR and lamina propria macrophages-derived mediators are able to induce intestinal secretion in vivo, probably via inhibition of protein phosphatase.     

非对称性二甲基精氨酸促进大鼠腹腔巨噬细胞诱导型一氧化氮合酶表达

目的观察非对称性二甲基精氨酸(asymmetric dimethylarginine,ADMA)对培养的大鼠腹腔巨噬细胞诱导型一氧化氮合酶(iNOS)表达的影响,探讨其在脂质沉积中的作用及机制。方法分组①正常对照组以PBS缓冲液与巨噬细胞共孵育,②氧化型低密度脂蛋白(oxLDL)组在巨噬细胞培养液中加oxLDL(50mg/L),③O A组在巨噬细胞培养液中加oxLDL(50mg/L)和ADMA(20μmol/L),④O A L组在培养液中加ox-LDL(50mg/L)、ADMA(20μmol/L)、L-Arg(1.2mmol/L)。以硝酸还原酶法和化学比色法分别测定培养液中一氧化氮(NO)含量与iNOS活力,以RT-PCR和Western Blotting分别检测iNOSmRNA和蛋白表达。结果①OxLDL组与O A组NO含量降低,iNOS活力升高,且以O A组明显,与正常对照组比较差异均有统计学意义(均P<0.05);NO与iNOS呈负相关(r=-0.697,P<0.05)。②O A组iNOS mRNA和蛋白表达增强,与正常对照组相比差异有统计学意义(P<0.05或P<0.01)。结论ADMA抑制大鼠腹腔巨噬细胞合成NO,增强iNOS基因与蛋白表达,可能是ADMA促使巨噬细胞转变为泡沫细胞、促进动脉粥样硬化发生发展的机制之一。     

神经胶质瘤中肿瘤相关巨噬细胞研究进展

肿瘤相关巨噬细胞（TAM）是异质性细胞群体,是肿瘤微环境中炎性细胞的主要成分。这些细胞在神经胶质瘤中部分源自中枢神经系统小胶质细胞和循环单核细胞,并与神经胶质瘤的血管生成、免疫抑制、肿瘤进展和侵袭有关。文章综述了TAM通过各种途径促进神经胶质瘤发展的潜在机制,为神经胶质瘤的靶向治疗提供了新的可能性。     

靶向肿瘤相关巨噬细胞治疗卵巢癌的研究进展

卵巢癌是致死率最高的妇科恶性肿瘤,免疫因素在卵巢癌的发生、发展中起重要作用。肿瘤相关巨噬细胞（tumor-associated macrophages,TAMs）是卵巢癌肿瘤微环境中重要的免疫细胞,可分泌多种细胞因子促进癌细胞增殖及血管生成并抑制肿瘤免疫,在卵巢癌进展中发挥重要作用。因此,靶向TAMs治疗卵巢癌成为目前的研究热点之一。其具体治疗策略包括：抑制TAMs的募集、增强TAMs的吞噬能力、消耗TAMs、将M2型TAMs去极化为M1型TAMs或抑制TAMs向M2型极化以及阻断TAMs与肿瘤细胞的相互作用。目前,已有诸多基础研究证实化疗药物、免疫检查点阻断剂、纳米药物和中药调节TAMs治疗或协同治疗对卵巢癌具有一定疗效。综述靶向TAMs治疗卵巢癌的原理及相关进展,以期为其进一步研究及临床应用提供参考。