Heat shock of rabbit synovial fibroblasts increases expression of mRNAs for two metalloproteinases, collagenase and stromelysin.

Two metalloproteinases, collagenase and stromelysin, are produced in large quantities by synovial fibroblasts in individuals with rheumatoid arthritis. These enzymes play a major role in the extensive destruction of connective tissue seen in this disease. In this study, we show that heat shock of monolayer cultures of rabbit synovial fibroblasts increases expression of mRNA for heat shock protein 70 (HSP-70), and for collagenase and stromelysin. We found that after heat shock for 1 h at 45 degrees C, the mRNA expression for HSP-70 peaks at 1 h and returns to control levels by 3 h. Collagenase and stromelysin mRNA expression is coordinate, reaching peak levels at 3 h and returning to control levels by 10 h. The increase in mRNA is paralleled by an increase in the corresponding protein in the culture medium. 3 h of heat shock at a lower temperature (42 degrees C) is also effective in inducing collagenase and stromelysin mRNAs. Concomitant treatment with phorbol myristate acetate (PMA; 10(-8) or 10(-9) M) and heat shock is not additive or synergistic. In addition, all-trans-retinoic acid, added just before heat shock, prevents the increase in mRNAs for collagenase and stromelysin. Our data suggest that heat shock may be an additional mechanism whereby collagenase and stromelysin are increased during rheumatoid arthritis and perhaps in other chronic inflammatory stress conditions.

Single step screening of monoclonal antibodies against interferon-gamma-induced surface molecules on human monocytes.

Interferon-gamma (IFN-gamma) activation of human monocytes in vitro results in enhanced phagocytosis and cellular cytotoxicity. These enhanced effector functions are attributable, at least in part, to increased expression of recognition molecules on the plasma membrane. In this article we report a rapid screening procedure for the primary selection of monoclonal antibodies (mAbs) which bind to cell surface molecules, the expression of which is increased or decreased by IFN-gamma. The procedure is based on flow cytometric analysis of mixed cell populations. Mouse mAbs were prepared using human monocytes cultured for 40 h with 400 U/ml of IFN-gamma as the immunogen. The hybridoma supernatants were screened using mixtures of six cell populations, some of which were pretreated with IFN-gamma for 40 h. Cells included in the mixture were chosen for their distinctive light scatter profiles. mAbs of interest were identified by preferential binding to monocytes and increased or decreased binding to monocytes treated with IFN-gamma. This procedure allowed us to screen several hundred clones per day, and to immediately eliminate mAbs that bound to B cells, T cells, neutrophils, and several cell lines. We selected ten mAbs which bound to surface molecules on monocytes that were modulated by IFN-gamma. Further characterization of five of the initial ten mAbs revealed that mAb gamma M phi 22.2 and mAb gamma M phi 197.1 bind to the high affinity Fc receptor for IgG (Fc gamma RI). mAb gamma M phi 28.3 appears to bind to a class II histocompatibility antigen and mAb gamma M phi 150.3 and mAb gamma M phi 195.18 appear to have binding patterns to human leukocytes and cell lines which are distinct from previously described mAbs. This rapid and specific procedure for screening mAbs has broad application for selecting mAbs that are specific for any given cell type and/or for surface molecules that are modulated by any cytokine and other hormone.

Microelectrode characterization of the basolateral membrane of rabbit S3 proximal tubule.

The purpose of this study was to characterize the basolateral membrane of the S3 segment of the rabbit proximal tubule using conventional and ion-selective microelectrodes. When compared with results from S1 and S2 segments, S3 cells under control conditions have a more negative basolateral membrane potential (Vbl = -69 mV), a higher relative potassium conductance (tK = 0.6), lower intracellular Na+ activity (ANa = 18.4 mM), and higher intracellular K+ activity (AK = 67.8 mM). No evidence for a conductive sodium-dependent or sodium-independent HCO3- pathway could be demonstrated. The basolateral Na-K pump is inhibited by 10(-4) M ouabain and bath perfusion with a potassium-free (0-K) solution. 0-K perfusion results in ANa = 64.8 mM, AK = 18.5 mM, and Vbl = -28 mV. Basolateral potassium channels are blocked by barium and by acidification of the bathing medium. The relative K+ conductance, as evaluated by increasing bath K+ to 17 mM, is dependent upon the resting Vbl in both S2 and S3 cells. In summary, the basolateral membrane of S3 cells contains a pump-leak system with similar properties to S1 and S2 proximal tubule cells. The absence of conductive bicarbonate pathways results in a hyperpolarized cell and larger Na+ and K+ gradients across the cell borders, which will influence the transport properties and intracellular ion activities in this tubule segment.

Recombinant mouse Bcl-2(1-203). Two domains connected by a long protease-sensitive linker.

Bcl-2 is a cytoplasmic integral membrane protein with potent anti-apoptotic activity but whose mechanism of action is poorly understood. The purpose of this paper was to obtain large amounts of soluble Bcl-2 protein for structural and functional studies. Mouse Bcl-2(1-203) (missing the COOH-terminal hydrophobic tail) was produced in bacterial inclusion bodies, solubilized in guanidine, and refolded by dialysis. The resulting protein was monomeric in nondenaturing solution and was active in protecting mouse T hybridoma cells from glucocorticoid-induced apoptosis. Refolded Bcl-2(1-203) showed no tendency to homodimerize by gel filtration or analytical ultracentrifugation. Limited proteolysis experiments identified a region between the BH3 and BH4 homology domains of Bcl-2(1-203) which was extremely susceptible to digestion by several common proteases, but not by a cell extract known to contain CPP-32-like (interleukin-1beta-converting enzyme family) protease activity. The protease-sensitive sites were located within a 50-residue stretch that contained most of the nonconserved and proline residues of Bcl-2(1-203). Trypsin-cleaved Bcl-2(1-203) eluted in the same position as the undigested protein on gel filtration in nondenaturing solution, indicating that the two portions of the molecule connected by the protease-sensitive region associate stably and noncovalently. The solution properties of Bcl-2(1-203) suggest that it consists of two noncovalently associated domains connected by a long protease-sensitive linker and that its structure is similar to that of Bcl-xL, which has been determined by x-ray and NMR analysis.

Multiple dimeric forms of human CD69 result from differential addition of N-glycans to typical (Asn-X-Ser/Thr) and atypical (Asn-X-cys) glycosylation motifs.

CD69 is expressed on the surface of all hematopoietically derived leukocytes and is suggested to function as a multipurpose cell-surface trigger molecule important in the development and activation of many different cell types. Human CD69 contains only a single consensus sequence for N-linked oligosaccharide addition within its extracellular domain (Asn-Val-Thr), yet exists as two distinct glycoforms that are assembled together into disulfide-linked homodimers and heterodimers. The molecular basis for human CD69 heterogeneity has remained elusive. In the current report we show that human CD69 glycoforms are generated before the egress of CD69 proteins from the endoplasmic reticulum to the Golgi and are synthesized under conditions where Golgi processing is inhibited, effectively ruling out the possibility that CD69 heterogeneity results from the differential processing of a single glycosylation site in the Golgi complex. Importantly, these data demonstrate that contrary to current belief, not one but two sites for N-glycan addition exist within the human CD69 extracellular domain and identify the second, "cryptic" CD69 N-glycan attachment site as the atypical Cys-containing glycosylation motif, Asn-Ala-Cys. The results in this study provide a molecular basis for human CD69 heterogeneity and show that multiple dimeric forms of human CD69 result from the variable addition of N-glycans to atypical and typical glycosylation motifs within the CD69 extracellular domain.

Binding of monomeric human IgG defines an expression polymorphism of Fc gamma RIII on large granular lymphocyte/natural killer cells.

Human PBL express one or more of the three classes of Fc receptors for IgG (Fc gamma receptors, I, II, and III). Each type of Fc gamma receptor has a characteristic binding pattern for isotypes of human and mouse IgG. Large granular lymphocyte/NK cells (LGL/NK cells) express the transmembrane form of Fc gamma RIII (Fc gamma RIIIa) on their surface, whereas polymorphonuclear neutrophils (PMN) express the glycosyl phosphatidyl inositol-linked receptor that is the product of the Fc gamma RIIIB gene. Fc gamma RIII has been reported to have low affinity for monomeric IgG, regardless of the cell type on which it is expressed. This study demonstrates specific and saturable binding of monomeric human IgG (M-IgG) to Fc gamma RIIIa on LGL/NK cells but not to Fc gamma RIIIb on PMN. M-IgG binding revealed an expression polymorphism between individuals, those with either high (12,000 to 15,000) or low (4,000 to 7,000) binding sites per cell. Both high and low M-IgG-binding LGL/NK cells bound hulgG1 and IgG3 but did not bind IgG2 or IgG4. The high/low Fc gamma RIIIA expression polymorphism correlated with the ability of an individual's LGL/NK cells to perform antibody-dependent cellular cytotoxicity of erythroid cell targets.

Distinct but dispensable N-glycosylation of human CD69 proteins.

Human CD69 is uniquely glycosylated at typical (Asn-X-Ser/Thr) and atypical (Asn-X-Cys) motifs, which represents the molecular basis for the formation of CD69 homodimers and heterodimers. Here we examined the importance of N-glycosylation for the assembly and intracellular transport of CD69 proteins using mutant CD69 molecules that specifically lack typical and atypical N-glycan attachment motifs. These studies verify the importance of Cys residues in atypical triplet sequences for N-glycan addition to human CD69 proteins in the endoplasmic reticulum (ER). In addition, these data demonstrate that monoglycosylated CD69 proteins (bearing N-glycans exclusively at atypical or typical sites) and aglycosylated CD69 molecules (lacking N-glycans) efficiently dimerize in the ER and have similar stability as wild-type CD69 molecules. Finally, these results show that CD69 proteins lacking atypical or typical N-glycan addition sites are transported to the plasma membrane.

Monoclonal antibodies that bind to distinct epitopes on Fc gamma RI are able to trigger receptor function.

The human monocyte and macrophage Fc receptor that binds human IgG with high affinity is a surface glycoprotein with a relative molecular mass of approximately 70 kDa. This receptor (Fc gamma RI) has been partially characterized using mAb 32 which binds outside the Fc binding domain of the receptor, but nonetheless triggers Fc receptor-dependent functions. In this study, we describe the properties of four new antibodies with specificity for Fc gamma RI. Based on additivity and cross-blocking studies, we conclude that two of these antibodies (mAb 22 and 44) define a third epitope which is distinct from the binding sites for both mAb 32 and the Fc portion of human IgG. Each Fc gamma RI-specific hybridoma was selected for stable sublines expressing high levels of mAb on the cell surface, and then tested for the ability of this surface mAb to trigger antibody-dependent cell cytotoxicity. All sublines were killed by human monocytes when used as targets in a 51Cr-release assay, whereas hybridomas specific for myeloid Ag other than Fc gamma RI were not killed. We conclude that Fc receptor function is triggered through binding to each of the three epitopes of Fc gamma RI that we have defined. These mAb will be useful for additional characterization of Fc gamma RI, and may, when incorporated into tumor-directed heteroantibodies, enhance tumor cell killing by human monocytes and macrophages.