Characterisation of macrophages infiltrating human mammary carcinomas.
Characterisation of macrophages infiltrating human mammary carcinomas.
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巨噬细胞浸润人乳腺癌的特征。
作者：
R. Steele;M. Brown;O. Eremin
众所周知，大多数恶性肿瘤都含有不同数量的巨噬细胞（Evans, 1977; Russell et al, 1981），但它们在人类乳腺癌中的存在却很少有文献记载。虽然Wood & Gollahon（1977）证实了乳腺肿瘤中存在携带Fc（IgG）受体的细胞，但一些研究者认为恶性细胞可以表达这种受体（Tonder & Thunold, 1973; Svennevig & Andersson, 1982）。形态学上，巨噬细胞在癌组织切片中不容易被描绘，在肿瘤细胞悬液中精确识别也同样困难。这就需要使用细胞标记物，在本研究中，巨噬细胞通过表达Fc（IgG）和C3受体以及具有HLA-DR抗原的能力来鉴定。为了更精确地表征这些细胞，还使用了与髓单细胞抗原反应的单克隆抗体（Kraft等人，1979）和与上皮膜抗原反应的多克隆抗体（Heyderman等人，1979）。本文研究了13例经组织学证实的原发性浸润性乳腺癌，临床局限于乳房和腋窝。手术切除的部分癌清除脂肪和筋膜，用组织培养基（TCM）洗涤。中药由RPMI1640 （Gibco, Europe）加10%热灭活胎牛血清、链霉素（100 ug ml-1）、青霉素G （100,000 iu ml-1）、0.7 G碳酸氢钠1-1和25mM HEPES缓冲液组成。肿瘤标本在中药中切块，在15 ml胶原酶（Sigma, Type I， 300 U ml -溶解在不含血清的中药中）中37°C孵育12小时，偶尔搅拌。细胞悬液经无菌纱布层过滤，中药洗涤6次，去除残留碎片和组织碎片。然后将细胞重悬于2 × 106mI-1的TCM中，使用相差显微镜评估细胞数量和活力（Eremin et al., 1982）。研究了四种表面标记物：IgG Fc部分和C3受体，HLA-DR抗原，由小鼠单克隆抗体BT 2/9鉴定，巨噬细胞相关抗原由小鼠单克隆抗体VEP-7鉴定。用ea - rotting法检测Fc（IgG）受体。简单地说，将牛红细胞(E)与低凝集但最佳剂量的兔IgG抗牛红细胞抗血清(a) （1:100 PBS）在室温（RT）下孵育45分钟（Eremin et al., 1976）。将致敏EA指示细胞（中药中2%悬液）与等体积的肿瘤细胞悬液（2 × 106个细胞ml1）混合并纺丝。将颗粒置于4°C静置30分钟，然后缓慢连续旋转1分钟重悬。C3受体通过EACrosetting反应被证实。将牛红细胞(E)与低凝集但最佳剂量的兔IgM抗牛红细胞抗体(a) （PBS中1:40）在4℃下孵育15分钟，然后与等体积的C5缺陷小鼠血清（补体固定试验稀释液中1:10）在37℃下孵育15分钟（Eremin et al., 1976）。然后将敏化的EAC指标用于如上所述的结花反应。建立适当的对照（热失活补体，EA-IgM），结果为阴性。中药中不存在Fc（IgM）活性（Eremin et al, 1982）。HLA-DR抗原和巨噬细胞相关抗原通过直接抗球蛋白凝集反应（DARR）检测，如前所述（Coombs et al., 1977）。在本实验中，用0.02%氯化铬偶联适当的抗体到胰蛋白酶处理的绵羊红细胞。后者在RT下缓慢旋转60min，在PBS中洗涤，在PBS中制成1%的悬浮液，并在4°C下保存。通过实验验证了耦合的有效性
It is well established that most malignant tumours contain a variable number of macrophages (Evans, 1977; Russell et al., 1981), but their presence in human breast cancer has been poorly documented. Although Wood & Gollahon (1977) demonstrated Fc(IgG) receptor-bearing cells in breast tumours, some investigators believe that malignant cells can express such receptors (Tonder & Thunold, 1973; Svennevig & Andersson, 1982). Morphologically, macrophages are not readily delineated in tissue sections of carcinomas, and in tumour cell suspensions precise identification is likewise difficult. This necessitates the use of cell markers, and in the present study, macrophages were identified by their ability to express receptors for Fc(IgG) and C3 and to possess HLA-DR antigens. In order to characterise these cells more precisely, a monoclonal antibody reactive with a myelomonocytic antigen (Kraft et al., 1979) and a polyclonal antibody reactive with epithelial membrane antigen (EMA) (Heyderman et al., 1979), were also employed. Thirteen women with histologically confirmed primary invasive mammary carcinoma, clinically confined to the breast and axilla were studied. A portion of the carcinoma removed at operation was cleared of fat and fascia and washed in tissue culture medium (TCM). TCM consisted of RPMI1640 (Gibco, Europe) with 10% heat-inactivated foetal calf serum, streptomycin (100 ug ml-1), penicillin G (100,000 i.u. ml1), 0.7 g sodium bicarbonate 1-1 and 25mM HEPES buffer. The tumour specimen was diced in TCM, and the pieces incubated in 15 mls collagenase (Sigma, Type I, 300 U ml1-dissolved in TCM without serum) at 37°C for 12 h with occasional agitation. The cell suspension was filtered through sterile gauze layers and washed six times in TCM to remove residual debris and tissue fragments. The cells were then resuspended in TCM at 2 x 106mI-1, and cell numbers and viability were assessed using phase contrast microscopy (Eremin et al., 1982). Four surface markers were studied: the receptors for the Fc portion of IgG and for C3, HLA-DR antigen, identified by the mouse monoclonal antibody BT 2/9, and a macrophage-associated antigen defined by the mouse monoclonal antibody VEP-7. Fc(IgG) receptors were detected by means of the EA-rosetting reaction. Briefly, ox erythrocytes (E), were incubated with a subagglutinating but optimal dose of rabbit IgG anti-ox erythrocyte antiserum (A) (1:100 in PBS) for 45min at room temperature (RT) (Eremin et al., 1976). The sensitised EA indicator cells (2% suspension in TCM) were mixed with equal volumes of the tumour cell suspensions (2 x 0l6 cells ml1) and spun down. The pellet was allowed to stand at 4°C for 30 min and then resuspended by slow, continuous rotation for 1 min. C3 receptors were demonstrated using the EACrosetting reaction. Ox erythrocytes (E) were incubated with a subagglutinating but optimal dose of rabbit IgM anti-ox erythrocyte antibody (A) (1:40 in PBS) for 15 min at 4°C and then with an equal volume of C5 deficient mouse serum (1:10 in complement fixation test diluent) for 15 min at 37°C (Eremin et al., 1976). The sensitised EAC indicators were then used in rosetting reactions as described above. Appropriate controls (heatinactivated complement, EA-IgM) were set up and found to be negative. Fc(IgM) activity was absent in TCM (Eremin et al., 1982). The HLA-DR antigen and the macrophage-associated antigen were detected by the direct antiglobulin rosetting reaction (DARR), as detailed previously (Coombs et al., 1977). In this assay, the appropriate antibody was coupled, using 0.02% chromic chloride to trypsin-treated sheep erythrocytes. The latter were slowly rotated at RT for 60min, washed in PBS, made up to a 1% suspension in PBS and stored at 4°C. The efficacy of the coupling was checked by a