Although calreticulin (CRT) is a major Ca2+-binding luminal resident protein, it can also appear on the surface of various types of cells and it functions as an immunopotentiating molecule. However, molecular mechanisms underlying the potent immunobiological activity of cell surface CRT are still unclear. In the present study, a recombinant fragment (rCRT/39–272) covering the lectin-like N domain and partial P domain of murine CRT has been expressed in Escherichia coli. The affinity-purified rCRT/39–272 assembles into homodimers and oligomers in solution and exhibits high binding affinity to various glycans, including carrageenan, alginic acids, and hyaluronic acids. Functionally, rCRT/39–272 is capable of driving the activation and maturation of B cells and cytokine production by macrophages in a TLR-4–dependent manner in vitro. It specifically binds recombinant mouse CD14, but not BAFFR and CD40. It is also able to trigger Ig class switching by B cells in the absence of T cell help both in vitro and in vivo. Furthermore, this fragment of CRT exhibits strong adjuvanticity when conjugated to polysaccharides or expressed as part of a fusion protein. Soluble CRT can be detected in the sera of patients with rheumatoid arthritis or systemic lupus erythematosus, but not in healthy subjects. We argue that CRT, either on the membrane surface of cells or in soluble form, is a potent stimulatory molecule to B cells and macrophages via the TLR-4/CD14 pathway and plays important roles in the pathogenisis of autoimmune diseases.

Although caltreticulin (CRT) is mainly a residential ER protein, it is also expressed on the membrane surface of various types of cells exhibiting multiple functions. We report here that intraperitoneal administration of a soluble recombinant CRT fragment (rCRT/39-272) led to a substantial decrease in delayed type hypersensitivity (DTH) responses in BALB/c mice and EAE in C57BL/6 mice. In the recall response against keyhole limpet hemocyanin (KLH) in vitro, draining lymph node cells from the rCRT/39-272-treated mice produced less IFN-γ but more IL-4 as compared with the cells from the control group. The immunomodulating effect of intraperitoneally administered rCRT/39-272 was attributed to anti-CRT Abs thereby induced, because, in passive transfer experiments, the CRTspecific antiserum could suppress DTH in BALB/c mice. B-cell-deficient μMT mice were not susceptible to rCRT/39-272-mediated DTH suppression. Furthermore, CRT appears on the surface of murine T cells soon after activation and remains detectable (at relatively low level) by flow cytometry for approximately 5 days in vitro. Anti-CRT Abs were able to inhibit AKT phosphorylation, proliferation, and cytokine production by activated murine T cells. We propose that cell surface CRT could play a role in the function of effector T cells and may be considered a target for immunological manipulation.Keywords: Anti-CRT antibodies r Calreticulin r Immunoregulation Supporting Information available online IntroductionCalreticulin (CRT) is a multifunctional glycoprotein of 46 kDa, and is most abundant in the ER of the cell [1][2][3][4][5][6]. It folds into three domains: (i) a lectin-like N domain, (ii) a proline-rich P domain, and (iii) a Ca 2+ -binding C-domain. CRT has well-recognized physiological roles in intracellular Ca 2+ storage and signaling and also as a molecular chaperone [1,5,6]. It has been detected in soluble form in the sera of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus [7,8]. We have shown that a recombinant CRT fragment 39-272 containing its partial NCorrespondence: Prof. Xiao-Ming Gao e-mail: xmgao@suda.edu.cn and P-domains (CRT/39-272) exhibits potent immunostimulatory activity and strong adjuvanticity [7], implying soluble CRT may be involved in immunopathological and inflammatory reactions leading to the development of RA and/or systemic lupus erythematosus. Moreover, CRT can be exposed at the membrane surface of various types of cells and exhibits multiple cellular functions as a surface molecule [6]. For example, cell-surface CRT (csCRT) mediates thrombospondin (TSP) triggered disassembly of focal contacts, exhibits antithrombotic effects, and inhibits melanoma cell spreading and angiogenesis [5,9,10]. Ligation of neuronal csCRT by complement C1q triggers increased levels of cellular * These authors contributed equally to this work. Eur. J. Immunol. 2012. 42: 2419-2430 reactive oxygen species [11]. There is also ample evidence showing that surface exposure of CRT enhances the immunogenicity of tumor cells and ...