A new monoclonal antibody, PM-2K, specifically recognizes tissue macrophages but not blood monocytes

A new monoclonal antibody, PM-2K, was raised against 24-h cultured human peritoneal macrophages. Immunohistochemically, PM-2K recognized most tissue macrophages in lymphoreticular organs such as the thymus, spleen, lymph node, and tonsil. Kupffer cells of the liver, alveolar macrophages, and macrophages in the interstitial tissue of the kidney, pancreas, and many other organs were also positively labelled. On the other hand, PM-2K failed to recognize blood monocytes, freshly isolated peritoneal macrophages, microglial cells, osteoclasts, and dendritic cells such as Langerhans cells, interdigitating cells, and follicular dendritic cells. In various pathological conditions, PM-2K labelled a wide variety of proliferating macrophages. Reaction products of PM-2K were observed by immunoelectron microscopy on the cytoplasmic membrane of cultured peritoneal macrophages. The molecular weight of the antigen recognized by PM-2K was determined to be 150 kD by Western blotting. As no cells other than macrophages were reactive with PM-2K, this antibody is considered to be very useful not only in the investigation of macrophage differentiation and maturation, but also in the diagnosis of various proliferative disorders of macrophages.     

Inhibition of murine macrophage IL-12 production by natural and synthetic DNA

DNA is a complex macromolecule whose immunological properties vary with sequence and structure. To determine whether DNA can inhibit immune responses, the effects of mammalian DNA and synthetic phosphodiester (Po) and phosphorothioate (Ps) oligonucleotides (ODNs) on IL-12 production were tested using murine macrophages. With bacterial DNA as a stimulant, calf thymus DNA and human placenta DNA blocked IL-12 production by splenic and bone marrow macrophages. A (dG)(30) Po ODN and all single-base Ps 30 mer ODNs were also effective inhibitors. The Ps ODNs also blocked IL-12 production induced by lipopolysaccharide (LPS) and a stimulatory Ps ODN. With the J774 cell line, single-base Ps ODNs inhibited IL-12 production induced by bacterial DNA, LPS, and a stimulatory Ps ODN. Together, these results indicate that DNA has inhibitory properties, suggesting that mammalian DNA could limit immune activation during inflammation and counteract the effects of bacterial DNA.     

Redistribution of fibroblasts and macrophages as micrometastases develop into established liver metastases

Fibroblastic tissue is an important component of malignant tumors, involved in the establishment of metastatic foci from micrometastases, and thought to prevent invasion of metastatic tumor cells into surrounding tissue. However, experimental models of fibrosis during the growth of micrometastasis into established metastases were not previously available. In the present paper, we performed immunohistochemical studies on experimental hepatic metastasis with colon 38 mouse colon carcinoma cells injected into syngeneic C57BL/6 mice. Early and late stages of metastatic nodules were examined for the distribution of endothelial cells, fibroblasts, and macrophages by the use of markers of these cells. One week after intrasplenic injection of colon 38 cells, micrometastases mainly appeared in the region of sinusoids accompanied with invasion of F4/80-positive Kupffer cells. Transitional metastases can be defined based on the histological appearance and intensive infiltration of both macrophages and fibroblasts. These transitional metastases were connected by protrusions of fibroblast-rich tissues co-localized with collagen-rich matrix and CD31-positive cells. This protrusion preceded fibrosis formation characteristics to established metastases associated with angiogenesis and segregation of tumor cells from host cells. Three stages can thus be classified during the development of hepatic metastasis in this syngeneic experimental system: micrometastasis, transitional metastasis, and established metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biological properties associated with the enhanced lung-colonizing potential in a B16 murine melanoma line grown in a medium conditioned by syngeneic Corynebacterium parvum-elicited macrophages

A previous study by our laboratory showed that the peritoneal murine Corynebacterium parvum-elicited macrophages released into their growth medium an activity which enhanced the ability of B16-F10 melanoma cells to form experimental metastases in the lung of syngeneic mice. In the present study, we used a clone of B16-F10 line (F10-M3 cells) to investigate whether the increase in lung-colonizing potential due to the pro-clonogenic activity released by C. parvum-elicited macrophages was associated with biological properties characteristic of a metastatic phenotype. We have found that the pulmonary retention, growth rate in lung parenchyma, invasiveness through Matrigel, adhesiveness to IL-1-activated endothelium and MHC class I expression were increased in F10-M3 cells stimulated by the macrophage pro-clonogenic activity. By using an in vitro experimental protocol, the enhancement of lung-colonizing potential in the stimulated melanoma cells turned out to be a transient phenomenon as was the increase of invasiveness through Matrigel and the higher expression of MHC class I antigens. In conclusion, the melanoma cells stimulated by the pro-clonogenic activity released by C. parvum-elicited macrophages showed changes in biological parameters which are relevant to metastatic diffusion. These changes appeared as a temporary phenomenon which sustains the view that the metastatic phenotype represents a transient biological character influenced by host factors. This revised version was published online in July 2006 with corrections to the Cover Date.     

小鼠肺纤维化模型中基质金属蛋白酶-9及其抑制剂表达水平的变化

目的 :研究小鼠博来霉素在肺纤维化模型中肺泡巨噬细胞(AM)分泌的基质金属蛋白酶 9(MMP 9)和基质金属蛋白酶抑制剂 1(TIMP 1)的表达随着时间的变化 ,观察肺纤维化中MMP 9和TIMP 1的表达是否存在失衡。方法 :以博来霉素诱导建立小鼠肺纤维化模型 ,采用HE染色观察肺部胶原沉积状况。选用抗CD6 8mAb ,采用微小免疫磁珠法分离纯化肺泡灌洗液中的AM ,用Hoechst 332 5 8染色AM ,在下光镜高倍视野计数法评估AM的纯度和活性 ,用EIA法检测AM上清液中MMP 9和TIMP 1的表达。结果 :肺组织切片HE染色显示小鼠肺部胶原沉积在 1、3、7、14、2 8d呈逐渐加重趋势。粗分离的AM纯度为 (82 .5± 2 .5 ) %。采用微小免疫磁珠法分离肺泡灌洗液中的AM纯度可达到 (99.3± 0 .7) % (P <0 .0 5 )。纯化后的细胞存活率为 (92 .5± 1.8) % ,与纯化前的 (92 .7± 2 .0 ) % ,相差不大 (P >0 .0 5 )。随着小鼠肺间质纤维化的进展 ,MMP 9的分泌量逐渐减少 ,而TIMP 1的表达量逐渐增加。结论 :在小鼠肺纤维化中AM分泌的MMP 9和TIMP 1之间存在失衡 ,表明基质降解系统受到抑制     

Type III hyperlipoproteinema with apolipoprotein E2/2 genotype in Japan

Limited information is available concerning type III hyperlipoproteinemia (HLP) in the Asian population. Therefore, clinical and biochemical characteristics of type III HLP were examined in 16 Japanese patients. Mean plasma triglyceride (TG) and total cholesterol (chol) levels were 381 mg/dl and 253 mg/dl, respectively, and the mean very low density lipoprotein (VLDL)-chol/plasma TG ratio was 0.27, which were lower than those reported in Western countries. Eighty percent of the patients had high plasma remnant-like particles (RLP)-chol levels above 50 mg/dl and a high RLP-chol/plasma TG ratio above 0.1. Twelve patients (75.0%) were obese. Seven patients (43.8%) had type 2 diabetes mellitus and four patients (25.0%) had impaired glucose tolerance. Six patients (37.5%) had coronary heart disease (CHD), but none had peripheral vascular disease or xanthomas. TG-rich lipoproteins from type III HLP patients with diabetes mellitus stimulated cholesteryl ester synthesis by human macrophages significantly (p < 0.001) more than those from type III HLP patients without diabetes mellitus. In conclusion, the Japanese type III HLP patients had lower plasma TG and total chol levels and a lower VLDL-chol/plasma TG ratio, but CHD was more common. The patients were characterized by a high frequency of obesity and/or glucose intolerance. The TG-rich lipoproteins from type III HLP patients with diabetes mellitus were more atherogenic.     

Characterization of a population of small macrophages in induced sputum of patients with chronic obstructive pulmonary disease and healthy volunteers

The inflammatory process in chronic obstructive pulmonary disease (COPD) is active mainly in the airways, but little is known about the properties of the inflammatory cells in this compartment. We have studied leucocytes in induced sputum of COPD patients compared to controls in order to uncover what types of macrophages might be involved in the disease. Sputum induction was performed by inhalation of nebulized sodium chloride solution. Leucocytes were isolated and stained with specific monoclonal antibodies for analysis in flow cytometry. Flow cytometry analysis revealed that a major portion of CD14+ macrophages in COPD has lower forward scatter, i.e. they are small macrophages. While in control donors these small macrophages accounted for 6.9% of all macrophages, the percentage of these cells in COPD was 45.7%. CD14 and HLA-DR expression was high on these small sputum macrophages while the large sputum macrophages expressed only low levels of these surface molecules, both in control donors and COPD patients. Small sputum macrophages of both control donors and COPD patients showed higher levels of constitutive tumour necrosis factor (TNF) compared to the large macrophages. TNF was inducible by lipopolysaccharide (LPS) preferentially in the small sputum macrophages in the control donors but there was no further induction in COPD patients. These data show that the small sputum macrophages are a major macrophage population in COPD and that these cells exhibit features of highly active inflammatory cells and may therefore be instrumental in airway inflammation in COPD.     

Technical improvements in the clotted plasma droplet MIF assay in vitro.

The migration inhibitory activity of culture supernatants of rat and human lymphocytes stimulated with PHA and Con A-Sepharose was tested on cell migration from clotted plasma droplets. This technique was improved by using homologous as well as heterologous plasma and fibrinogen solutions for suspending the migratory cells, automatic micropipettes for performing the technique and purified cell populations as target. The effects of calcium chloride and of the cell concentration in the plasma droplets on the migration indices obtained in the MIF assay were tested.     

Increased expression of adhesion molecules (ICAM-1 and LFA-1) on alveolar macrophages from asthmatic patients

In the airways inflammation observed in asthma, activated macrophages are present in increased numbers. Adhesion molecules are required for the cell: cell contacts between leukocytes and endothelial cells or other leukocytes, and they are induced by inflammatory stimuli. We studied the expression of two adhesion molecules (ICAM-1 and LFA-1) on alveolar macrophages recovered by bronchoalveolar lavage from 11 normal subjects and 13 asthmatic patients by using immunocytochemistry. Two specific monoclonal antibodies were used, and the reaction was revealed by the alkaline phosphatase-antialkaline phosphatase (APAAP) method. The percentage of cells expressing ICAM-1 or LFA-1 was significantly increased in asthmatic patients, as compared with normal subjects ( P < 0.001 and P < 0.002, respectively; Mann-Whitney U test), and there was a significant correlation with the percentage of cells expressing both markers in asthma ( P < 0.03, Spearman rank test). This study highlights the importance of macrophages in the inflammation of asthma and suggests that macrophage interactions with other cells play a role in this inflammation.     

Participation of hematopoietic stem cells in formation of the immune response

The effect of different doses of sheep's red blood cells (SRBC) on endogenous colony formation and immunogenesis was studied in CBA and C57BL mice. The absolute and relative numbers of antibody-forming cells (AFC) was shown to increase with an increase in the dose of SRBC from 2×10⁷ to 2×10⁸, and to decrease with a dose of 2×10⁹ in mice of both lines. With an increase in the dose of antigen, the number of endogenous splenic colonies (ESC) of hematopoietic cells also increased. Interlinear differences in the number of ESC persisted. It is suggested that the mechanism of stimulation of the hematopoietic series is based on a unique type of blockade of the reticuloendothelial system by foreign red blood cells, leading to increased proliferative activity of the stem cells.Laboratory of Regulation of Immunopoiesis, Institute of Clinical and Experimental Medicien, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 4, pp. 330–332, April, 1979.