Amala Guha

Expression of a Negative Regulator of Human Erythropoiesis by Fluidized Lymphocyte Plasma Membranesa

Regulation of hematopoiesis by paracrine molecules occurs in vitro. In some cases, hematopoietic paracrine factors have been localized to the plasma membrane of accessory cells. We have purified a unique integral membrane glycoprotein from normal human B cells that functions in vitro as a paracrine factor whose activity is directed toward erythroid progenitor cells. This factor is also spontaneously exfoliated from the cell surface as a component of extracellular vesicles. Analysis of the lipid and protein compositions and membrane lipid order of these extracellular vesicles reveals them to be biochemically distinct and more fluid than their parent membranes. Evidence in nonhematopoietic culture systems indicates that cell membrane function may be altered by modifying membrane fluidity. In an effort to accelerate growth factor release, plasma membranes of B cells were fluidized by incubation with an emulsion of Liposin II, phosphatidylcholine, and phosphatidylethanolamine. Fluidity assessed by steady-state fluorescence polarization was reduced in lipid-treated cells. Exfoliation was 3-4-fold higher from lipid-treated cells relative to untreated cells. Unexpectedly, a negative signal for burst-forming unit-erythroid (BFU-E) proliferation was expressed in membranes, in shed extracellular vesicles, and in supernatants of medium conditioned by the fluidized cells. Purification of the inhibitor is under way. The data are consistent with the hypothesis that accessory cell plasma membranes may positively or negatively regulate erythroid differentiation, depending upon the exchange of cholesterol and phospholipids between plasma membrane and ambient lipid pools.

Synthesis of prostaglandin E by peritoneal macrophages from NZB/W mice

Peritoneal macrophages from NZB/W (murine lupus) mice spontaneously produce less prostaglandin E (PGE) than peritoneal macrophages from immunologically normal mice. Reduced PGE synthesis is seen as early as 2 months of age and becomes more profound as disease progresses. It is suggested that impaired production of PGE by peritoneal macrophages from NZB/W mice may account in part for abnormal macrophage function observed in these animals.

Inhibition of major histocompatibility complex class I antigen shedding up-regulates the surface expression of class I antigens on the lymphocyte cell surface.

Interferon-gamma induces the expression of major histocompatibility complex class I and class II gene products. Moreover, the density of MHC antigens present on the lymphocyte surface is regulated by exfoliation of the plasma membrane. To probe the cellular mechanisms involved in IFN-gamma-induced alteration of MHC antigen expression, we measured the effects of IFN-gamma on the rate of MHC antigen shedding and the biosynthesis of H-2Dd. Balb/c splenic lymphocytes were surface-iodinated with 125I and incubated in the presence and absence of up to 1000 U/ml IFN-gamma, or they were metabolically labeled with [35S]methionine with or without 500 U/ml IFN-gamma. Radioiodinated or 35S-labeled H-2Dd was quantified by immunoprecipitation of H-2Dd from detergent lysates of radiolabeled cells that were incubated with the appropriate antibody for 4-20 h at 37 degrees C. Monoclonal antibody 34-5-8 was employed as a specific probe for H-2Dd. Loss of radioiodinated H-2Dd from the cell surface was diminished by 75-90% at 12 h in tests of lymphocytes continuously cultured with IFN-gamma (compared to control, p less than 0.05). In contrast, the biosynthetic rate was unaffected during the initial 10 h of incubation. The net result of these changes was the early appearance of an increase in H-2Dd on the cell surface. This result was in accordance with data obtained by phenotyping the untreated and treated cells using double-antibody staining methods and fluorescence-activated cell sorter analysis. Our results suggest that IFN-gamma induces MHC expression by initially retarding the exfoliation of MHC antigens from the lymphocyte surface. Delayed effects on MHC expression may be, on the other hand, mediated by increased antigen biosynthesis.

Induction of Erythropoiesis by MHC-Mediated Cognate Interactions between B- and T-Cells

We have previously shown that the expression of membrane burst-promoting activity (mBPA), an erythropoietic cytokine, by B-lymphocytes is augmented by the addition of allogeneic effector cells to the B-cells. Here, we have examined immune mechanisms involved in the induction/promotion of erythropoiesis as assessed by the capacity of autologous and allogeneic peripheral blood lymphocytes to augment burst-forming unit-erythroid (BFU-E) in normal human bone marrow cells in vitro. Preincubation of mBPA-expressing human B-cells with monoclonal antibodies to major histocompatibility complex (MHC) antigens, abrogated erythropoietic activity of both autologous and allogeneic lymphocytes, suggesting that MHC antigens play a role in regulating the expression of the erythroid growth factor. Inhibition of BFU-E proliferation was also evident when antibodies to MHC class-I or class-II antigens were added directly to marrow culture. Furthermore, addition of anti-CD4 antibody to the cultures of PBL and autologous target BM cells markedly reduced erythroid proliferation induced by PBL. By contrast, anti-CD8 and control (UPC-10) monoclonal antibodies had no effect. These results provide evidence that MHC-mediated cognate interactions between T- and B-lymphocytes may participate in the control of erythropoiesis, either directly or by modulating mBPA function.

Inhibition of Hematopoiesis in Vitro with the Lym‐1 Monoclonal Antibody

Lym-1 is a monoclonal antibody that was produced and purified by Epstein et al.‘ Nuclei from the Burkitt’s lymphoma Raji cell line were isolated, and BALB/c mice were immunized. Splenocytes were obtained and fused with myeloma NS-1 cells, and hybridomas were formed. Lym-1 is an IgG,, monoclonal antibody, purified from tissue culture supernatant by protein-A-Sepharose affinity chromatography, that recognizes a polymorphic variant of the HLA-DR antigen present on the surface of normal and malignant B cells. Similar determinants may also be expressed by human hematopoietic progenitor cells2J or by critical hematopoietic accessory When radioconjugated Lym-1 is administered to patients with B cell lymphomas, shrinkage of lymphoma frequently occurs. However, cytopenias, hypoplastic bone marrows, and aplastic anemia may also occur, findings that cannot be attributed to the low dose of radiation administered.J Because of the hematologic toxicities, none of the patients was able to receive a second course of treatment. One patient developed irreversible aplastic anemia, resulting in death. Lym-1 and its F(ab’), fragment (Lym-1-F(ab’),) were directly conjugated to fluorescein isothiocyanate (FITC; fluorochrome: protein ratios of 1.5: 1 and 3: 1, respectively) by standard methods. In addition, Lym-1 was biotinylated by standard methods. Purified Lym1, biotinylated Lym1, Lym1 -FITC conjugate, and Lym1-F(ab’),-FITC conjugate were used to determine: (1) the effects of Lym-1 on hematopoiesis in vitro; (2) the capacity of Lym1 to immunoabsorb hematopoietic progenitor cells by panning and by attachment to avidin-Sepharose beads; and (3) the staining by Lym1 of human peripheral blood and bone marrow light-density mononuclear cells. The effects of Lym-1 on hematopoiesis were determined by its addition to human marrow culture.6 Dose-dependent suppression of colony formation in presence of complement was observed for all colony types, except for mature erythroid progenitors. Interestingly, this pattern of responsiveness is identical to that described for HLA-DR expression. Moreover, Lym1 has also been shown to exhibit complement-mediated cytotoxicity, but not direct cytotoxicity, for Raji cells.’ Inhibition is evident at concentrations achievable in viw (2-25 pg/ml serum at 1 h after administration of Lym-1). These results suggest that Lym1 recognizes determinants present on either hematopoietic progenitor/stem cells or critical hematopoietic accessory cells that promote hematopoietic proliferation/differentiation in vitro.