James S. Peacock

Lectins and antibodies as tools for studying cellular interactions

Specific interactions between multiple cell types are critical for a variety of processes central to the development, homeostasis and immune defense of multicellular organisms. Studies designed to elucidate how cells communicate through physical encounters have exploited exogenously supplied factors to bypass intrinsic recognition mechanisms and facilitate cellular conjugation. In this review, we compare the relatively nonspecific agglutinating properties of lectins and the selective cell targeting capabilities of antibodies and bispecific antibody constructs for studying cell-cell interactions in immunobiology. In addition, we discuss a novel system for inducing cellular interactions which closely resembles native receptor-mediated conjugation. In this system, surrogate receptors promote specific cell-cell interactions without hindering endogenous receptor-ligand interactions at the cell-cell interface which may be important in mediating physiologic cellular responses.

The use of palmitate-conjugated protein A for coating cells with artificial receptors which facilitate intercellular interactions

A method is described in which palmitate-conjugated protein A (pal-protein A) incorporated onto cell membranes is used to anchor antibodies on the surface of cells. Pal-protein A was stabilized on the cell surfaces via insertion of the hydrophobic palmitate moieties into the phospholipid bilayer of the plasma membrane. This membrane-associated pal-protein A retained its binding affinity for the Fc region of rabbit immunoglobulin G (IgG) molecules. Using this system, cells can be coated with antibody (Ab) molecules of a desired specificity that can serve as artificial receptors for soluble or cell surface antigens (Ags). In this report, we show that A22.E10 T hybridoma cells coated with rabbit anti-mouse IgG (RaMIgG) exhibited increased adhesiveness to surface IgG positive mouse B cells. A high level of heterotypic intercellular conjugation was observed in cells coated with specific artificial receptors as compared to cells coated with nonspecific artificial receptors.

Protein and lipid lateral diffusion in normal and Rous sarcoma virus transformed chick embryo fibroblasts

We measured the lateral diffusion of the fluorescent lipid analogue dioctadecylindocarbocyanine iodide (DiI) and of membrane glycoproteins labeled with tetramethylrhodamine (TRITC) succinyl concanavalin A (SConA) via fluorescence photobleaching recovery (FPR) at selected times during a temperature downshift experiment on transformation-defective temperature-sensitive (td-ts) Rous sarcoma virus (RSV) NY68-transformed chicken embryo fibroblasts (CEF) and on identically treated CEF and RSV-transformed CEF. There were no significant differences in the lateral diffusion in DiI at any of the times measured. The lateral diffusion of TRITC-SConA on the RSV-transformed CEF, (1.32 +/- 0.12).10(-10) cm2 s-1, was approximately two times faster than that observed in normal CEF, (0.61 +/- 0.06).10(-10) cm2 s-1. In the cells undergoing RSV NY68-mediated transformation, TRITC-SConA diffusion increased over a 24-h period from a value comparable to that observed in normal CEF, (0.72 +/- 0.13).10(-10) cm2 s-1 to a value comparable to the RSV-CEF transformed cells, (1.74 +/- 0.20).10(-10) cm2 s-1. All diffusion measurements reported were made at the permissive temperature for RSV-NY68 (35 degrees C) unless stated otherwise. The changes in the lateral diffusion of TRITC-SConA occurred between the fifth and twelfth hour of the downshift course and could be associated with cytoskeletal disruption and/or fibronectin degradation, both known to occur at this time in RSV-transformed cells. To assess the contribution of extracellular matrix (ECM) degradation, SConA mobility was measured in normal and RSV-transformed cells treated with trypsin. This treatment increased SConA mobility approximately 4-fold in the normal cells relative to untreated controls and only 2-fold in the RSV-CEF transformed cells. No significant difference in SConA mobility between trypsinized spherical normal and transformed cells was apparent.

Effects of interleukin 4 on neonatal B lymphocyte tolerance

Perturbation of antigen receptors on mouse neonatal B cells by rabbit antimouse IgM antibody was shown to inhibit cell proliferation in response to the B cell mitogen lipopolysaccharide. When these antibody-inactivated cells were challenged with lipopolysaccharide in the presence of the helper T cell product interleukin 4, a strong proliferative response was observed. Interleukin 4 alone did not cause proliferation of the antibody-treated B cells. Pretreatment with interleukin 4 did not prevent neonatal B cell inactivation by the antibody. Our results show that neonatal B cells inactivated directly through their antigen receptors can be reactivated by the combined signals of interleukin 4 and lipopolysaccharide.

Triamcinolone acetonide inhibits lymphocyte differentiation in B cells decorated with artificial antigen receptors

We examined the effects of triamcinolone acetonide (TA) on T cell independent antigen-induced differentiation of human B cells. Purified human B cells artificially decorated with palmitate-conjugated monoclonal IgA antibody specific for 2,4-dinitrophenyl differentiated polyclonally when challenged with optimum concentrations of dinitrophenyl-derivatized polymerized flagellin. This B cell response was reduced by the synthetic corticosteroid TA at a concentration of 10(-6) M. This suggests that TA can inhibit in vitro B lymphocyte differentiation independent of T cells.

Sodium pyruvate inhibits the spontaneous release of 51Cr from RBC in chromium release assays

This report describes the utility of sodium pyruvate for markedly decreasing the spontaneous release of chromium-51 from RBC in long term chromium release assays. This method can be used to increase the sensitivity and duration of cytotoxicity assays which use chromium-51-labeled RBC as targets.

Anti-Ig inactivation of the ch31 lymphoma model for immature B cell inhibits its ability to process pigeon cytochrome C

We wished to determine whether tolerized cells are able to process and present antigen based on our hypothesis that tolerized B cells be unable to function in the normal capacity as antigen-presenting cells if they are to remain the tolerant state. Results in this study show that the ability of the murine lymphoma model for immature B cells, CH31, to process pigeon cytochrome c was greatly down-regulated when cultured in the presence of rabbit anti-mouse IgM. In contrast, the same anti-IgM treatment had no significant effect on the antigen-presenting cell function of the lymphoma model for mature B cells, CH112. Presentation of CNBr-cleaved fragments of pigeon cytochrome c by either CH31 or CH12 cells was not affected by the antibody treatment. Furthermore, CH31 cells pre-incubated with pigeon cytochrome c were not subject to the anti-IgM inhibition of the antigen presentation. These observations suggest that pertubation of surface immunoglobulin molecules on CH31 immature B cells causes down-regulation of their antigen-processing machinery.

Monovalent Fab fragments of D7.5 monoclonal antibody activate intracellular Ca2+ mobilization and secretion of cytolytic factors by thymus cells.

Previous studies have identified two mouse monoclonal antibodies, D7.5 and G1.4, that each can cause secretion of cytolytic factors from natural killer (NK) cells and resting T cells in humans, rats, and hamsters. Toward elucidation of the molecular identity and the transmembrane signaling mechanism of the antibody‐defined trigger molecules, we investigated 1) their surface density, 2) the crosslinking requirement for signaling, and 3) their ability to mobilize intracellular calcium ions. Equilibrium binding studies using monovalent Fab fragments of D7.5 showed the presence of approximately 20,000 trigger molecules per thymus cell and an antibody dissociation constant of 1.8 x 10‐6 M. Thymus cells incubated in the presence of either intact or Fab fragments of D7.5 acquired the ability to lyse Yac‐1 cells. Furthermore, the incubation media of thymocytes that were treated with either intact or Fab fragments of D7.5 contained effector‐cell derived factors that could lyse Yac‐1 cells. Flow cytometry measurements of changes in intracellular free calcium concentration ([Ca2+]i) in thymocytes. A significantly elevated level of [Ca2+]i was observed at 60s after antibody addition and this response, which required the external calcium source, increased continuously during the 30 min incubation. Furthermore, dbcAMP plus theophylline did not alter the ability of D7.5 to induce calcium mobilization suggesting that the antibody‐defined trigger may not use the signaling pathway involving breakdown of phosphatidylinositol.