Optimization of cellular ELISA for assay of surface antigens on human synoviocytes.

The cellular enzyme-linked immunosorbent assay (CELISA) permits assay of cell surface antigens on intact fixed cells. Using a monolayer of cells as the solid phase, the CELISA offers an inexpensive alternative to flow cytometry. In addition, this protocol has the decided advantages of miniaturization (small numbers of cells) and ease of replication (the 96-well cluster plate format). In efforts to optimize CELISA for detecting surface antigens on human fibroblastlike synoviocytes, the authors found that cell number, serum proteins, choice of culture plate, pipetting technique and fixatives may all impact the results of the CELISA.

Prolactin-like protein-B: heterologous expression and characterization of placental and decidual species.

Prolactin-like protein-B (PLP-B) is a member of a family of proteins expressed by the rat placenta and/or decidua with characteristics similar to prolactin (PRL). In this report, we present the heterologous expression and characterization of PLP-B. Recombinant PLP-B heterologously expressed in Chinese hamster ovary cells exhibited similar immunoreactive and electrophoretic characteristics with PLP-B produced by rat placental and decidual tissues. N-terminal sequencing verified the identity and purity of the recombinant PLP-B species and the site of cleavage of the signal peptide from the mature secreted PLP-B species. Polyclonal antibodies were generated to the recombinant PLP-B and used for Western blot and immunocytochemical analyses. Recombinant and native PLP-B migrated as a doublet at 30-31 kDa in SDS-PAGE under reducing conditions. Treatment of recombinant and native PLP-B with N-glycanase accelerated their electrophoretic mobility, indicative of their glycoprotein nature. PLP-B was localized exclusively to decidual cells in the developing deciduum and spongiotrophoblast cells in the placental junctional zone. The level of PLP-B protein expression dramatically declined prior to parturition. Potential PRL-like biological actions of PLP-B were also investigated. PLP-B bound weakly to ovarian and liver PRL receptors and did not stimulate the proliferation of lactogen-dependent Nb2 lymphoma cells. In conclusion, recombinant PLP-B possesses characteristics similar to native decidual and placental PLP-B and may represent a hormone/cytokine that has important modulatory actions during the establishment of pregnancy and the initiation of parturition.

Placental lactogen-I variant utilizes the prolactin receptor signaling pathway.

Placenta lactogen-I variant (PL-Iv) is a member of a family of proteins expressed by the rat placenta with characteristics similar to prolactin (PRL). In this report, we present the molecular cloning, chromosomal localization, and heterologous expression of PL-Iv. Nucleotide sequence analysis of the PL-Iv cDNA clone predicted a precursor protein of 223 amino acids, including a 28-amino acid signal sequence. The PL-Iv gene was localized to chromosome 17 of the rat genome, which also carries other members of the PRL gene family. PL-Iv heterologously expressed in Chinese Hamster ovary (CHO) cells exhibited similar immunoreactive and electrophoretic characteristics with PL-Iv produced by the rat placenta. N-terminal sequencing verified the identity and purity of the recombinant PL-Iv species and the site of cleavage of the signal peptide from the mature secreted PL-Iv species. Recombinant PL-Iv was shown to bind to ovarian and liver PRL receptors, stimulate the proliferation of Nb2 lymphoma cells, and activate Jak2. Each of these actions is consistent with PL-Iv utilizing the PRL receptor signal transduction pathway.

Developmental regulation of a pregnancy-specific oligosaccharide structure, NeuAcalpha2,6GalNAcbeta1,4GlcNAc, on select members of the rat placental prolactin family.

Successful pregnancy is dependent upon an array of signaling proteins secreted by the trophoblast cells of the placenta. Among these is a group of proteins related to pituitary prolactin, known as the prolactin/growth hormone family. These proteins are expressed at specific times during gestation and synthesized in distinct trophoblast cell types in the rat placenta. We report here that select members of this family, prolactin-like protein (PLP-A), PLP-B, PLP-C, decidual/trophoblast PRP, and placental lactogen I variant, only which are expressed in the spongiotrophoblast, late in rat placental development bear Asn-linked oligosaccharides terminating with NeuAcalpha2,6GalNAcbeta1,4GlcNAcbeta-R. This reflects the concurrent expression of these prolactin/growth hormone family members with the peptide-specific beta1,4GalNAc-transferase and an alpha2,6-sialyltransferase, which can add sialic acid to terminal beta1,4-linked GalNAc. We have determined that at least one of the prolactin-like proteins, PLP-A, is recognized by the protein-specific GalNAc-transferase. The presence of NeuAcalpha2, 6GalNAcbeta1,4GlcNAcbeta-R on only a limited number of glycoproteins synthesized by the spongiotrophoblasts between mid gestation and birth reflects the need for both the GalNAc-transferase and the peptide recognition determinant for efficient addition of GalNAc. Thus, expression of the GalNAc-transferase and specific members of the prolactin/growth hormone family is developmentally regulated in the rat placenta, suggesting a physiological role for the terminal NeuAcalpha2,6GalNAcbeta1,4GlcNAcbeta-R sequence on Asn-linked oligosaccharides of these proteins.

Proinflammatory cytokines enhance human synoviocyte expression of functional intercellular adhesion molecule-1 (ICAM-1).

We determined the ability of proinflammatory cytokines to enhance ICAM-1 (CD54) expression on, and PBMC adhesion to, human synoviocytes. Surface molecules were characterized by cell ELISA and by flow cytometry. Adhesion of PBMC to synoviocyte monolayers was measured by direct counting or by colorimetric staining. Most cytokines upregulated ICAM-1 expression (IL-1 beta > TNF alpha > IFN-gamma >> PDGF-bb, IL-6), but not GM-CSF or TGF beta. A similar concentration-dependent increase was observed for synoviocytes derived from patients with rheumatoid or osteoarthritis. Kinetic studies of ICAM-1 expression differed among several cytokines: an early rise with IL-1 beta or TNF alpha stimulation, a gradual increase with IFN-gamma, a transient increase with PDGF-bb, and a plateau with IL-6. Adhesion of PBMC to synoviocytes was increased by IL-1 beta or TNF alpha and reduced by MAb to CD54 or CD18. Increased synoviocyte adhesiveness may promote interactions with infiltrating inflammatory cells.

Analysis of elevated serum interleukin-6 levels in rheumatoid arthritis: correlation with erythrocyte sedimentation rate or C-reactive protein.

The purpose of this study was to determine whether selected antirheumatic drugs would suppress elevated circulating interleukin-6 (IL-6) levels in patients with rheumatoid arthritis (RA). The 267 patients who enrolled in a double-blind randomized protocol received placebo, naproxen (1500 mg/day), or prinomide (1500 mg/day) for up to 16 weeks. Serum samples from 143 of the patients completing the trial and from 135 normal donors were analyzed by quantitative sandwich enzyme-linked immunosorbent assay for IL-6 concentrations. A mean normal IL-6 value was determined to be 3 pg/ml (95th percentile value = 10 pg/ml). IL-6 levels at baseline for the patients with RA were significantly higher than those for control subjects (p < 0.0001). Elevated IL-6 levels (> 10 pg/ml) at baseline were found in 80% of subjects with RA (median = 36 pg/ml, range 12 to 403). For patients with elevated levels of either IL-6, C-reactive protein (CRP), or erythrocyte sedimentation rate (ESR) at baseline, initial median values of IL-6, CRP, and ESR were compared with those from the final visit for each treatment group. There was no significant decrease in IL-6 levels with treatment. Median CRP levels decreased significantly, from 1.9 to 0.8 mg/dl (p = 0.002), as did median ESR (37 to 34 mm/hr, p = 0.013), only in the prinomide-treated group.