We have previously reported (O'Garra, A. et al., Int. Immunol. 1990, 2:821) that murine B lymphomas and purified normal peritoneal B cells produce interleukin (IL) 10. We now show that this production of IL 10 B cells correlates with the presence of Ly-1 (B-1) B cells, in both normal and diseased mice. Using a semi-quantitative modification of the polymerase chain reaction, we show that IL 10 expression is detectable in peritoneal B cells but only becomes apparent in splenic B cells of aged mice of which a high proportion are Ly-1+. Furthermore, the expression of IL 10 is constitutive in splenic B cells from mice carrying the Ly-1+ BCL1 lymphoma. Since IL 10 is a potent regulator of in vitro immune function, its production by Ly-1 lineage B (B-1) cells raises the possibility that this subset of B cells may regulate their own development and/or the function of other immunocompetent cells.

We have examined a panel of murine Ly-1+ B lymphomas and purified normal murine peritoneal B cells separated into subsets on the basis of expression of the Ly-1 surface antigen, for their ability to produce cytokines. Where possible, we have used a combination of cytokine detection methods in order to compensate for differences in sensitivity and specificity, and the possibility of inhibitors masking an activity. All the lymphomas tested were shown to constitutively express TGF-beta and CSIF/IL-10. In addition, varying levels of IL-6, TNF-alpha and TNF-beta, and G-CSF, were demonstrable in most of the lymphomas, and variants of one lymphoma (CH12) additionally produced varying levels of IL-3, IL-4, and GM-CSF. FACS purified normal Ly-1+ and Ly-1- peritoneal B cells, were also shown to express RNA encoding CSIF/IL-10, IL-6, TNF-alpha and TNF-beta, and very low levels of G-CSF, following stimulation with LPS. These data were supported by the detection of IL-6 and CSIF/IL-10 in supernatants from LPS-stimulated Ly-1+ and Ly-1- B cells using specific immunoassays. None of the lymphomas or B cell preparations produced IL-1 alpha, IL-2, IL-5, IL-7, or IFN-gamma. The purity of our normal B cell populations was assessed by phenotypic analysis on the FACS and also by the disappearance of certain mRNA transcripts after purification, e.g. CD4, c-fms, GM-CSF, and IFN-gamma, most of which could be detected in LPS-stimulated total peritoneal cell populations. This suggested that our B cell purification method had reduced, to a level undetectable in our assays, contaminating T cells (CD4), macrophages (c-fms, GM-CSF), and NK cells (IFN-gamma). Absence of IL-3, IL-4, IL-5, and GM-CSF expression by LPS-stimulated Ly-1+ and Ly-1- B cells reduced the concern that contaminating peritoneal mast cells could account for the observed cytokine production. We therefore believe our data provide strong support for production of a subset of cytokines by LPS-stimulated normal B cells. Both the Ly-1+ B lymphomas and normal Ly-1+ and Ly-1- B cells appear capable of expressing IL-6, TNF-alpha, TNF-beta, and CSIF/IL-10.(ABSTRACT TRUNCATED AT 400 WORDS)

The most remarkable feature of the immune response is its ability to generate cells and antibodies specifically reactive with an enormous variety of foreign antigens while normally avoiding the production of autoreactive elements. An accumulating body of data demonstrates that a distinct lineage of B cells (1), the Ly-1 + B cell subset, does not display this feature, but rather is associated with the production ofautoreactive antibodies (2). We are unaware of autoimmune pathology directly demonstrated to be the result of an Ly-1 + B cell-Ig product; however, Ly-1+ B cell frequency is elevated in some autoimmune strains of mice (3). In addition to their association with autoreactive antibody, Ly-1 + B cells have been shown to have unique growth potential (1, 4); to produce most of the serum IgM found in normal, unimmunized animals (4); and the Ig repertoire of the subset may be restricted (4, 5). These findings imply that this subset has a role in normal physiology, and perhaps autoimmune pathology. A B cell subset has been identified in humans that bears the homologous marker Leu-1/T1 (CD5) (6). A similar functional role for CD5+ B cells in man has been proposed (7); human CD5+ B cells have been shown to produce autoreactive antibody (8, 9) and to be present at elevated frequency in rheumatoid arthritis (10).Normal mice produce antibodies that are reactive with protease-treated autologous erythrocytes (BrMRBC) 1 (11) ; cells producing such antibodies are present in unimmunized mice at very high frequencies, as first demonstrated by Cunningham (12) . We and others (13)(14)(15) have shown that at least some anti-BrMRBC plaqueforming cells (PFC) recognize the polar headgroup of the common membrane phospholipid, phosphatidyl choline (PtC). Most splenic anti-BrMRBC PFC have been

Most, if not all, autoantibodies specific for bromelain-treated mouse erythrocytes recognize the common membrane phospholipid, phosphatidyl choline (PtC). Anti-PtC antibodies are produced by 5%-15% of CD5+ Ly-1 B cells of normal unimmunized mice, but not by detectable numbers of conventional CD5- B cells. At 1 week of age PtC-specific B cells are undetectable but then increase dramatically over the next 3 to 4 weeks to reach adult numbers. We report here that PtC-specific Ly-1 B cells in B10.H-2aH-4bp/Wts mice predominantly express either of two heavy and kappa chain variable (V) region gene combinations. In addition, the sequence and length of DH genes are conserved among cells expressing the same V gene combination, and the V kappa-J kappa junctions of one group involve unusual splice sites. Preferential V gene rearrangement models are insufficient to explain the DH and V kappa-J kappa junctional sequences or the delayed appearance of this specificity, and so they cannot solely account for the high frequency of PtC-specific cells. These characteristics are more consistent with antigen selection. We therefore attribute the frequent use of the two V region gene combinations to selection for cells that express them and conclude that the expressed V gene repertoire of Ly-1 B cells in adult mice is influenced by antigen selection. Apparently, there is no selection for mutant anti-PtC antibodies of higher affinity during the formation of the Ly-1 B repertoire because the V region genes expressed by PtC-specific cells are unmutated. Our findings are consistent with an important, germ line-encoded function for the immunoglobulin products of these gene combinations.

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