Mansfield P J, Suchard S J
Department of Pediatrics, University of Michigan, Ann Arbor 48109.
J Immunol. 1994 Nov 1;153(9):4219-29.
The presence of the extracellular matrix protein thrombospondin (TSP) at sites of tissue injury or inflammation may promote monocyte migration to these sites and play a central role in their eventual differentiation into tissue macrophages. Previously, we have shown that TSP promotes neutrophil adhesion and migration, and primes for oxidant generation. To examine the effect of TSP on monocyte motility, we conducted chemotaxis assays in modified Boyden chambers. TSP was chemotactic for monocytes, with a maximal response at 200 to 500 nM TSP. Checkerboard analysis confirmed that migration was directional. mAb C6.7, against the distal COOH terminus of TSP, inhibited chemotaxis, demonstrating specificity and indicating that the chemotactic activity resides in the COOH terminus. Consistent with the mAb data, the COOH-terminal 140-kDa proteolytic fragment of TSP was chemotactic for monocytes, whereas the NH2-terminal heparin-binding domain was inactive. A synthetic peptide containing the sequence CSVT, derived from the type I repeats of TSP, was also chemotactic. Thus, two different sites on the COOH terminus of TSP are capable of stimulating monocyte chemotaxis. Pertussis toxin, but not cholera toxin, completely inhibited TSP-mediated chemotaxis, suggesting the involvement of GTP-binding proteins. TSP bound to polycarbonate filters stimulated monocyte haptotaxis, with a maximal response at 4 pmol. The directional nature of this motility was confirmed by checkerboard analysis. Monocyte haptotaxis was inhibited by two different mAbs recognizing distinct sites on the COOH terminus. As with chemotaxis, the 140-kDa fragment, but not the heparin-binding domain, contained the haptotactic activity. The CSVT-containing synthetic peptide also promoted monocyte haptotaxis. But, in contrast to chemotaxis, neither pertussis toxin nor cholera toxin inhibited TSP-mediated haptotaxis, suggesting the involvement of a different signal transduction pathway. mAbs against GPIV, beta 1, beta 3, or alpha v integrins did not affect monocyte chemotaxis or haptotaxis, ruling out the involvement of these receptors. These results indicate that TSP is likely to play an important role in monocyte recruitment to an inflammatory or injury site.
细胞外基质蛋白血小板反应蛋白（TSP）在组织损伤或炎症部位的存在可能会促进单核细胞向这些部位迁移，并在其最终分化为组织巨噬细胞的过程中发挥核心作用。此前，我们已经表明TSP可促进中性粒细胞的黏附和迁移，并引发氧化剂的产生。为了研究TSP对单核细胞运动性的影响，我们在改良的博伊登小室中进行了趋化性分析。TSP对单核细胞具有趋化作用，在200至500 nM TSP时反应最大。棋盘分析证实迁移是定向的。针对TSP远端COOH末端的单克隆抗体C6.7抑制了趋化作用，证明了其特异性，并表明趋化活性存在于COOH末端。与单克隆抗体数据一致，TSP的COOH末端140-kDa蛋白水解片段对单核细胞具有趋化作用，而NH2末端肝素结合域则无活性。一个包含源自TSP I型重复序列的CSVT序列的合成肽也具有趋化作用。因此，TSP的COOH末端上的两个不同位点能够刺激单核细胞趋化作用。百日咳毒素而非霍乱毒素完全抑制了TSP介导的趋化作用，提示GTP结合蛋白参与其中。结合在聚碳酸酯滤膜上的TSP刺激单核细胞进行接触趋化，在4 pmol时反应最大。这种运动性的定向性质通过棋盘分析得到证实。单核细胞接触趋化受到两种识别COOH末端不同位点的不同单克隆抗体的抑制。与趋化作用一样，140-kDa片段而非肝素结合域具有接触趋化活性。包含CSVT的合成肽也促进了单核细胞接触趋化。但是，与趋化作用不同，百日咳毒素和霍乱毒素均未抑制TSP介导的接触趋化，提示涉及不同的信号转导途径。针对GPIV、β1、β3或αv整合素的单克隆抗体不影响单核细胞趋化或接触趋化，排除了这些受体的参与。这些结果表明TSP可能在单核细胞募集到炎症或损伤部位中起重要作用。
