Isolation and function of a human endothelial cell C1q receptor

Area of Science:

• Immunology
• Cell Biology
• Biochemistry

Background:

• Cultured human endothelial cells (EC) bind C1q in a time- and dose-dependent manner, with approximately 5.2 x 10^5 binding sites per cell.
• Previous work demonstrated that C1q binding to EC induces the attachment of IgM-containing immune complexes (IC).

Purpose of the Study:

• To isolate and characterize the putative C1q receptor (C1qR) from human endothelial cells.
• To develop monoclonal antibodies (mAbs) against C1qR for further functional studies.
• To investigate the role of C1qR in the interaction between C1q, immune complexes, and endothelial cells.

Main Methods:

• Affinity chromatography using C1q-Sepharose to isolate C1qR from human umbilical cord EC membranes.
• Detection of C1qR activity by its ability to inhibit the lysis of EAC1q in C1q-deficient serum.
• Purification and molecular weight determination of C1qR using QAE-A50 chromatography and HPLC gel filtration.
• Generation of IgM monoclonal antibodies (mAbs) D3 and D5 against purified C1qR.
• Western blot analysis using anti-C1qR mAb D3.
• Functional assays measuring the binding of radiolabeled C1q to EC in the presence of mAbs.

Main Results:

• C1qR was isolated from 1-5 x 10^9 human umbilical cord EC.
• Purified C1qR exhibited an apparent molecular weight of approximately 60 kDa (55-62 kDa unreduced, 64-68 kDa reduced).
• Monoclonal antibodies D3 and D5 were generated; D3 recognized a 54-60 kDa protein band in EC and polymorphonuclear leukocyte membranes.
• Anti-C1qR mAb F(ab')(2) fragments inhibited C1q binding to EC in a dose-dependent manner, while intact mAbs enhanced C1q binding.
• The study supports a hypothesis where IgM-IC binding to C1q, followed by C1 activation and dissociation, leads to C1q-IC interaction with EC-C1qR.

Conclusions:

• The C1q receptor (C1qR) on human endothelial cells has been successfully isolated and characterized.
• Monoclonal antibodies against C1qR provide tools to study its function in C1q binding and immune complex interactions.
• The findings suggest a mechanism for how C1q-IC may interact with endothelial cells during primary immune responses via C1qR.