Allotype-specific immunoregulation of autoantibody production by host B cells in chronic graft-versus host disease

A chronic graft-vs-host (GVH) reaction induced in nonautoimmune mice by the transfer of Ia-incompatible spleen cells results in a syndrome that closely resembles SLE in the spectrum of autoantibodies and immunopathology. We have utilized Ia- and Igh-congenic strains to study the immunoregulation of autoantibody-producing B cells in this model. We have found that the autoantibodies are produced almost entirely by the host B cells. The transferred donor B cells contributed neither to the autoimmune response nor to the total serum Ig, with rare exceptions. The donor cell population did, however, exert an Igh allotype-specific immunoregulatory effect on the host B cells. For example, in allotype-heterozygous recipients, the autoantibodies were preferentially made by those host cells that expressed the donor allotype, whereas those host B cells that expressed nondonor allotype were relatively suppressed. In allotype-homozygous recipients, the donor cells frequently suppressed the host IgG2a allotype completely. This suppression sometimes prevented the IgG antichromatin response, although in other cases the response occurred with the use of a different isotype. In a final set of experiments, a chronic GVH reaction was induced in which both the donors and the recipients were Igh allotype heterozygous and yet differed at Ia. In this case, no donor influence on allotype should be expected; yet the IgG2a autoantibodies were clearly skewed toward the b allotype. These results show that host B cells play a unique role in the GVH autoimmune syndrome. In addition, they are immunoregulated in allotype-specific manners, some of which presumably involve interaction with donor T cells.     

Increased autoantibody production by NZB/NZW B cells in response to IL-5

We previously demonstrated that B cells from NZB/NZW but not nonautoimmune mice secrete high levels of autoantibodies in response to factor(s) derived from type 2 Th cell (Th2) clones. Supernatants from type 1 Th cell clones, which contain a different set of lymphokines, were not stimulatory. In the present experiments, we attempted to define the active Th2 factor(s) and to better understand the cellular basis for the hyperresponsiveness. In response to optimal concentrations of supernatant (Th2-Sup), B cells from 3-mo-old NZB/NZW mice produced up to 40-fold greater amounts of IgM anti-DNA compared with unstimulated B cells, whereas BALB/c B cells produced levels only slightly above background. Although Th2-Sup contained large amounts of IL-4, comparable concentrations of rIL-4 alone did not stimulate NZB/NZW B cells. Furthermore, a blocking anti-IL-4 mAb did not prevent Th2-Sup-stimulated autoantibody production. Th2-Sup was fractionated by HPLC, and the stimulatory factor(s) was found in fractions known to contain IL-5 (also known as B cell growth factor II). Indeed, a highly purified preparation of IL-5 reproduced the effects of Th2-Sup by stimulating NZB/NZW B cells to produce high levels of IgM anti-DNA antibodies while enhancing production by nonautoimmune cells only slightly. In limiting dilution studies, NZB/NZW compared with BALB/c spleens contained a three- to four-fold greater frequency of DNA-specific B cells that were responsive to IL-5. Together, the results suggest a potential role for IL-5 in the pathogenesis of NZB/NZW autoimmune disease.     

IL-4 promotes Stat6-dependent survival of autoreactive B cells in vivo without inducing autoantibody production

Persistent cross-linking of hen egg lysozyme (HEL)-specific B cell membrane Ig (mIg) in double transgenic mice that express soluble HEL as a self Ag (HEL-Ig mice) decreases B cell mIgM expression, responsiveness, and life span. Because in vitro treatment with IL-4 inhibits T cell apoptosis through a Stat6-independent mechanism, increases mIg expression, and suppresses activation-induced B cell death, we studied IL-4 effects on B cell mIg expression, survival, and Ab secretion in Stat6-sufficient and deficient HEL-Ig mice. IL-4 treatment nearly normalized B cell number and greatly increased the percentage of mature B cells in HEL-Ig mice, but failed to normalize mIgM expression or spontaneous LPS-induced IgM secretion. IL-4 effects on B cell survival and maturation were CD4(+) T cell independent, but Stat6 dependent, and did not involve receptor editing. IL-4 had to be present while B cells were generated to have a detectable effect on autoreactive B cell survival; however, the survival of B cells generated in the presence of IL-4 was substantially increased even after IL-4 was withdrawn. These observations suggest that: 1) activation-induced B cell death and anergy are independent processes; 2) B cells that survive to maturity develop increased resistance to Ag-induced deletion; and 3) IL-4 promotes B and T cell survival through different mechanisms.     

Studies on the uptake of exogenous phosphoglycerides by Escherichia coli B cells

The uptake of exogenous lipids by Escherichia coli B was studied under various conditions. Lysophosphoglycerides were absorbed more readily than diacyl analogues when sonicated dispersions of a single lipid were used. When these same lipids were admixed with coliform lipids and dispersed as vesicles, the uptake of lysophosphoglyceride diminished and was approximately equal to that of diacyl analogues. Neutral lipids such as diglyceride, fatty acid, and cholesterol were also absorbed when admixed and dispersed as vesicles with coliform lipids. The uptake of lysophosphoglyceride was stimulated slightly by all divalent cations tested except Mg2+; monovalent cations were ineffective. Uptake was accompanied by conversion of lysophosphoglyceride to diacyl analogues. In the presence of Ca2+, lysophosphatidylethanolamine also formed a more polar lipid product yet unidentified. The uptake of lipid did not cause release of 3H label into the medium from cells that had been grown in [3H]acetate-containing medium. Also, the [3H]phosphoglyceride content of such labelled cells remained constant. Thus the uptake process did not involve exchange of membrane lipid with the medium or an enhanced hydrolysis of endogenous lipids, but it represented a net gain of lipid by the cell. The uptake did not seem to involve a stable adsorption of lipid at the surface of the cell as could be judged from electron microscopic examination of the cells after incubation; the cell surfaces were devoid of adsorbed vesicle or liposomal types of structures and did not display evidence of expansion. [3H]Cholesterol-[32P]phospholipid mixtures were taken up without a change in isotopic ratio. This result together with the other evidence presented indicate a net uptake of exogenous lipid by a process likely involving fusion.     

Analysis of H2-O influence on antigen presentation by B cells

HLA-DM (DM; in mouse H2-DM) promotes the exchange of MHC class II-associated peptides, resulting in the accumulation of stable MHC class II-peptide complexes. In naive (but not germinal center) B cells, a large part of DM is tightly associated with HLA-DO (DO; in mouse H2-O), but the functional consequence of this association for Ag presentation is debated. Here, we have extended previous studies by examining the presentation of multiple epitopes after Ag internalization by fluid phase endocytosis or receptor-mediated uptake by membrane Ig (mIg) receptors. We find that the effects of H2-O are more complex than previously appreciated; thus, while only minor influences on Ag presentation could be detected after fluid phase uptake, many epitopes were substantially affected after mIg-mediated uptake. Unexpectedly, the presentation of different epitopes was found to be enhanced, diminished, or unaffected in the absence of H2-O, depending on the specificity of the mIg used for Ag internalization. Interestingly, epitopes from the same Ag did not necessarily show the same H2-O dependency. This finding suggests that H2-O may control the repertoire of peptides presented by B cells depending on the mIg-Ag interaction. The absence of DO/H2-O from germinal center B cells suggests that this control may be released during B cell maturation.     

Autoregulation of B cell growth by an immunoglobulin M autoantibody

This study documents the autoregulation of B cell growth by an IgM autoantibody. This autoantibody is secreted by B lymphocytes upon stimulation by polyclonal activators and is identified by its potent inhibition of B cell growth induced by these agents (endotoxin, Fc fragment of human gamma-globulin, Mycoplasma bovirhinis, and anti-mouse Ig). The autoantibody specifically affects B cells: there was no effect on mitogen- or antigen-induced T cell proliferation and of responses to interleukin 1, interleukin 2, or interferon. Nonspecific effects were excluded by the ineffectiveness of serum and myeloma IgM. Also, IgG-IgM immune complexes were excluded. The binding specificity of the IgM autoantibody is not yet defined but appears to be a B cell surface structure distinct from membrane Ig. The autoantibody constitutes a ubiquitous, autoregulatory influence on B cell growth, which may be an important component in physiologic and pathologic states of B cell homeostasis.     

In vitro production of anti-histone antibodies by spleen cells from young autoantibody negative NZB/NZW mice

Anti-histone antibodies (AHA) are spontaneously produced in NZB/NZW mice as part of their autoimmune disease. IgM AHA are usually not detected until after 4 mo of age, and older female mice switch to the production of IgG AHA. We studied the in vitro production of AHA by spleen cells from young (less than or equal to 12-wk-old) NZB/NZW mice. Despite the absence of elevated serum AHA activity, spleen cells from these mice demonstrated marked spontaneous autoantibody production in culture. In kinetic studies, little in vitro production was detectable after 1 day of culture, and maximal accumulation occurred on day 5. Elevated AHA production was apparent by cells from 2-wk-old NZB/NZW mice, and an age-dependent increase in autoantibody production was also noted. Only AHA of the IgM class were detected in cultures of young spleen cells. The in vitro production of IgM AHA in culture was T cell dependent, depletion of T cells resulting in a 70 to 90% reduction in production, which was corrected by the readdition of T cells. In cultures where both IgM AHA and total IgM secretion were measured, a much greater T cell dependence for AHA production was apparent. The requirement for T cells could also be partially replaced by factors present in concanavalin A supernatant. AHA secretion was induced by lipopolysaccharide by using cells from both NZB/NZW and non-autoimmune mice. Although production was greater with NZB/NZW cells, the difference was much less than that for spontaneous production. Thus, AHA-secreting cells that are dependent on in vitro T cell help are present in young NZB/NZW mice. These studies may help define the mechanisms responsible for selective autoantibody secretion in lupus-like disease.     

B cells amplify IFN-gamma production by T cells via a TNF-alpha-mediated mechanism

Aside from being the precursors of the Ab-secreting cells, B cells are engaged in other immune functions such as Ag presentation to T cells or cytokine production. These functions may contribute to the pathogenic role of B cells in a wide range of autoimmune diseases. We demonstrate that B cells acquire the capacity to amplify IFN-gamma production by CD4 and CD8 T cells during the course of the Th1 inflammatory response to Toxoplasma gondii infection. Using the two following different strategies, we observed that B cells from T. gondii-infected mice, but not from naive mice, induce higher IFN-gamma expression by splenic host T cells: 1) reconstitution of B cell-deficient mice with B cells expressing an alloantigen different from the recipients, and 2) adoptive transfer of B and T cells into RAG-/- mice. In vitro assays allowing the physical separation of T and B cells demonstrate that Ag-primed B cells enhance IFN-gamma production by T cells in a contact-dependent fashion. Using an OVA-transgenic strain of T. gondii and OVA-specific CD4 T cells, we observed that the proinflammatory effect of B cells is neither Ag specific nor requires MHCII expression. However, TNF-alpha expressed on the surface of B cells appears to mediate in part the up-regulation of IFN-gamma by the effector T cells.     

The influence of high ambient glucose level on the production of pericellular glycosaminoglycans by cultured endothelial cells

The 14C-acetate metabolic labeling of glycosaminoglycans (GAGs) was used to investigate the effect of high glucose level on the production of hyaluronic acid (HA), heparan sulphate (HS), chondroitin sulphate (CS) and dermatan sulphate (DS) by human immortalized umbilical vein endothelial cells. It is demonstrated that 30 mM glucose decreased the accumulation of HS and increased the accumulation of CS and DS in the cell layer, pericellular matrix and conditioned medium in 48 h of incubation. The modulation of the overall metabolism of sulphated GAGs by high glucose is in contrast to the observed redistribution of HA from the conditioned medium to the pericellular matrix of endothelial cells. The preincubation at 30 mM glucose increased also the attachment of hyaluronidase-treated endothelial cells to HA-coated surface and had no effect on the cell attachment to poly-D-lysine, indicating the alterations of CD44 binding to immobilized HA. The treatment of endothelial cells with p-nitrophenyl-beta-D-xylopyranoside, which inhibits the coupling of CS to the core protein, attenuated high glucose-induced pericellular HA accumulation and decreased cell attachment to HA-coated surface. It is supposed the implication of CD44-related CS in the accumulation of pericellular HA by endothelial cells exposed to high glucose level.     

The influence of composite traits on genotype by environment relations

Summary In breeding for multiple trait value functions, the existence of genotype-by-environment interaction effects can vastly complicate the designation of optimum sets of genotype-environment pairings into Target Populations of Environments. In this paper it is shown that even in the absence of any changes in genotypic ranking over environments on a trait-by-trait basis, it is possible to generate changes in genotypic ranking in value in different environments. This is shown to be true even for linear value functions in a case example in pine breeding.Published as paper No. 9570 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA