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We have designed and expressed bivalent small immune proteins (SIP) based on scFv fragments connected through a short linker of four amino acids to the CH3 domain of the human immunoglobulin gamma 1 H-chain. Three different versions have been designed and expressed in mammalian cells. In one construct a cysteine residue was included in the last amino acid of the flexible 15-amino acid long linker connecting the V(L) and V(H) domains, thus creating a disulphide bond stabilized molecule. A version with a shorter (five amino acids) V(L)/V(H) linker was also produced and shown to be efficiently assembled and secreted. All three SIPs form dimers retaining their antigenic specificity in Western blotting and having a comparable functional affinity (avidity) as determined by ELISA.

SUMMARYAlthough production of chemokines by vascular endothelial cells has been documented, there is only limited information regarding the expression of chemokines by the lymphatic endothelium. Here we used lymphatic endothelial cells (LEC) derived from experimentally induced murine lymphangiomas to investigate the pattern of chemokine expression by these cells. Histological analysis of the lymphatic hyperplasia revealed the presence of leucocytes in the tissues surrounding the lesions, suggesting the presence of chemoattractant activity. A functional chemotactic assay on human polymorphonuclear cells and on puri®ed subpopulations of murine leucocytes using culture supernatants from LEC primary cultures con®rmed the presence of chemoattractant activity. The identity of different cytokines of the CXC, CC and C subfamilies was investigated by reverse trancriptase±polymerase chain reaction on total endothelial cell RNA. Ampli®ed fragments corresponding to KC, IP10, Mig-1, BCL, MIP-2, SLC, RANTES, MCP-1, C10, and Lptn were obtained, and con®rmed by Southern blot and sequencing. In contrast, MIP-1a, MIP-1b, and MIP1g were not detected. Higher levels of expression were revealed by Northern blot analysis for Mig-1, MCP-1 and C10. The lymphatic endothelium-restricted production of these chemokines was also con®rmed by in situ hybridization. The presence of high C10 mRNA expression levels in LEC was particularly unexpected, because the production of this molecule has been previously identi®ed only in cells of the haematopoietic lineage. These observations represent the ®rst detailed analysis of chemokine production by lymphatic endothelial cells and may account, in part, for the mechanism of leucocyte recruitment into the lymphatics, and of lymphocyte recirculation within the lymphatic system.

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