Barker J N, Jones M L, Swenson C L, Sarma V, Mitra R S, Ward P A, Johnson K J, Fantone J C, Dixit V M, Nickoloff B J
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109-0602.
J Immunol. 1991 Feb 15;146(4):1192-7.
Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin.
在几种不同的炎症性皮肤病中，单核细胞会在表皮以及真皮 - 表皮交界处积聚。为了确定人类表皮角质形成细胞是否会产生一种特定的趋化因子，导致单核细胞在这些解剖部位积聚，研究人员使用人类外周血单核细胞作为靶细胞，对经16小时培养的角质形成细胞条件培养基中的单核细胞趋化活性进行了检测。稀释分析显示，在经干扰素 - γ（100 U/ml）处理的角质形成细胞培养基中，单核细胞呈定向迁移（最大FMLP反应的80%），这比干扰素 - γ本身或未处理的角质形成细胞活性高出10倍。凝胶过滤层析显示，这种活性在分子量为12.5 kDa的分子标志物之前洗脱。阻断研究表明，针对单核细胞趋化和激活因子（MCAF）的兔多克隆抗体可抑制所有单核细胞趋化，抑制率超过80%。用35S - 半胱氨酸/甲硫氨酸对角质形成细胞进行代谢标记，孵育16小时后，用相同的抗MCAF抗体对培养基进行免疫沉淀。仅在经干扰素 - γ（100 U/ml）处理的角质形成细胞培养基中，检测到MCAF为12 kDa和9 kDa的蛋白双峰。同时用干扰素 - γ和肿瘤坏死因子 - α（250 U/ml）孵育可导致MCAF蛋白产量增加。通过Northern印迹分析，MCAF mRNA在角质形成细胞中组成性表达，仅在存在干扰素 - γ时上调。肿瘤坏死因子 - α、白细胞介素 - β、转化生长因子 - β和佛波酯对MCAF mRNA没有正向或负向影响。这些研究表明，人类表皮角质形成细胞在被干扰素 - γ激活后会产生具有生物学活性的MCAF，干扰素 - γ也是诱导单核细胞与角质形成细胞之间黏附所必需的细胞因子。角质形成细胞衍生的MCAF可能在调节皮肤单核细胞迁移中起重要作用，也可能负责将与单核细胞/巨噬细胞具有许多表型特征的朗格汉斯细胞和真皮树突状细胞募集到皮肤中的解剖位置。
