No abstract

Objective. To analyze the molecular and functional characteristics of a soluble form of the granulocytemacrophage colony-stimulating factor receptor a chain (sGM-CSFRa), and analyze transcript expression in immune cells and the cellular constituents of rheumatoid arthritis synovial tissue.Methods. We amplified, cloned, and expressed the sGM-CSFRa and transmembrane form of the receptor (tmGM-CSFRa) from complementary DNA derived from a human myelomonocytic tell line. Competitive polymerase chain reaction assays were developed to determine the absolute and relative amounts of tmGMCSFRa versus sGM-CSFRa message synthesized by various cell lines and tissues.Results. sGM-CSFRa transcripts were detected in bone marrow, monocytehacrophages (cultured in GM-CSF), rheumatoid synovial tissue, and rheumatoid synovial tissue T cell lines, and represented the preDr. VonFeldt

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important in many immune and inflammatory processes. GM-CSF binds to specific cellular receptors which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design, and such design depends on a molecular understanding of ligand-receptor interactions. One approach to dissecting out critical intermolecular interactions is to develop analogs of specific interaction sites of potential importance. Monoclonal antibodies have been employed for these purposes in prior studies. Here we present application of recombinant antibody technology to the development of analogs of a site on GM-CSF bound by a neutralizing anti-GM-CSF monoclonal antibody. Polyclonal antisera with high titer neutralizing activity against human GM-CSF were developed in BALB/c mice. Purified immunoglobulins were prepared and used to immunize syngeneic mice. Anti-anti-GM-CSF was developed which demonstrated biological antagonist activity against GM-CSF-dependent cellular proliferation. RNA was extracted from spleen cells of mice with biologically active anti-anti-GM-CSF, cDNA synthesized, and polymerase chain reaction performed with primers specific for murine kappa light chain V regions. Polymerase chain reaction products were cloned into the pDABL vector and an expression library developed. This was screened with anti-GM-CSF neutralizing mAb 126.213, and several binding clones isolated. One clone (23.2) which inhibited 126.213 binding to GM-CSF was sequenced revealing a murine kappa light chain of subgroup III. Comparison of the 23.2 sequence with the human GM-CSF sequence revealed only weak sequence similarity of specific complementarity determining regions (CDRs) with human GM-CSF. Structural analysis revealed potential mimicry of specific amino acids in the CDR I, CDR II and FR3 regions of 23.2 with residues on the B and C helices of GM-CSF. A synthetic peptide analog of the CDR I was bound by 126.213, specifically antagonized GM-CSF binding to cells and blocked GM-CSF bioactivity. These studies indicate the feasibility of using recombinant antibody libraries as sources of interaction site analogs.

While many investigators have examined V gene usage by the clonotypic T-cell receptor (TCR) in rheumatoid arthritis (RA) joints, few have reported on arthritic controls. We compared TCR alpha-chain V gene usage in knee synovial tissue specimens from 9 RA and 5 osteoarthritis (OA) patients. There was no significant difference in the number of V gene families used in RA compared with OA synovium. However, there was an increased prevalence of V alpha 28, V alpha 10, V alpha 17, and V alpha 18 and under representation of V alpha 15 in RA compared with OA synovium. Of these, V alpha 28 was also recently described by us as being present in RA synovial tissue early in the course of disease. V alpha 28 associated J region usage, and N-regional diversity was surveyed in T-cell receptors from additional rheumatoid synovial tissue T-cell populations and normal peripheral blood. Oligoclonality was observed in 6/10 rheumatoid specimens either by direct sequencing or where three or more molecular clones were sequenced, compared with 0/5 normal PBMCs. The oligoclonal populations included 2/3 cell lines stimulated with interleukin-2 (IL-2) alone. Several novel J regions were observed, with some recurrent residues observed at N-region positions. These data indicate an increased prevalence of certain TCR V region families in RA versus OA synovium, and suggest an antigen-driven expansion of V alpha 28-expressing T cells in RA synovium.