Relationship between the synthesis of autoimmune antibodies and the formation of clusters of B,T and APC cells during the syngeneic mixed lymphocyte reaction in BALB/c and NZB mice: A technique for isolation of the spleen autoimmune compartment of non-immunized pathogen-free mice

Cells from the spleens of non-immunized mice were cultured in horizontal tubes, rotating very slowly around their long axis. Under these conditions, the flux speed gradient of the cell suspension near the tube walls greatly increased the chances of cells coming into contact with one another. Mixed clusters of B, T and APC cells were soon found adhering firmly to the walls of the tube; cluster formation leveled off after about 3 h. The clustered cells were easily separated from those remaining in suspension and constituted a particular cell compartment comprising a maximum of 20–30 % of the total.B cells from this compartment, cultured in complete medium for 48 h, almost exclusively produced IgM antibodies. Antibodies reacting with self antigens were so numerous in the culture medium that it is likely all IgM were self antibodies.That the clusters obtained under these conditions constituted a compartment of autoimmune cells is supported by previous work which showed that 20–30 % of spleen cells secrete IgM antibodies almost exclusively.Cluster formation as a function of age was compared in NZB mice which are used as a model of lupus erythematosus, and in BALB/c mice which never manifest self-immune pathology. The number of cells found in clusters per whole spleen increased exponentially with age in NZB mice and linearly in BALB/c mice. The production of autoimmune antibodies as a function of age also increased exponentially for NZB mice and linearly in BALB/c mice, which provides further striking support for the hypothesis that the clusters formed constitute the autoimmune comportment.     

Loss of lymphocyte chalone activity in mice with autoimmune disease.

Lymphocyte chalone from the spleens of old BALB/c, young BALB/c and young NZB mice caused significant suppression of the proliferative response of BALB/c and NZB spleen cells to T and B mitogens, whereas lymphocyte chalone from old NZB spleen did not suppress. Lymphocyte chalone from young and old NZB mice was tested using different ages of NZB/NZW responding spleen cells; at all ages concanavalin A- and lipopolysaccharide-induced proliferation was suppressed less by the chalone from old NZB mice than from that of young NZB mice. The responding NZB/NZW cells were suppressed equivalently at all ages studied. The basis for the loss of lymphocyte chalone activity in old NZB mice remains unknown; however, it appears likely that this event has a role in the disturbance of the negative feedback control system which contributes to NZB autoimmune disease.     

Secondary antibody response of overtly autoimmune NZB mice

Coombs' positive NZB mice, 13–18 months old, showed an active secondary response to immunization with sheep erythrocytes as determined by direct and indirect spleen haemolytic antibody plaque formation. The magnitude and pattern of response were essentially similar to those of old, non-autoimmune BALB/c mice for time intervals of 7, 15 or 25 days between primary and secondary stimuli, although peak NZB plaque formation was delayed by 1–2 days in the case of the 25-day time interval. These results were in marked contrast to the previously observed depression of primary immune capacity in overtly autoimmune NZB mice. It is speculated that upon priming formation of memory cells occurs at a relatively normal rate at the expense of cells usually involved in early primary antibody production.     

Bone marrow cells from young and old New Zealand black mice can reconstitute B lymphocytes in severe combined immunodeficient recipients

The formation of B lymphocytes in young New Zealand Black (NZB) mice proceeds at an accelerated rate, resulting in a deficiency of B lineage progenitors in mice of 15 weeks of age and older. Multiple studies have indicated that intrinsic defects in B lineage cells as well as in the hemopoietic microenvironment in which they develop contribute to these cellular abnormalities. To determine whether the B-cell hyperactivity observed in young mice could be observed in a normal environment, bone marrow cells from young (4 weeks or less) NZB donors were transplanted into Severe Combined Immunodeficient (SCID) mice that have a marked deficiency of lymphocytes but an apparently normal hemopoietic microenvironment. Engraftment of donor lymphoid cells can occur without pretransplant conditioning regimens, thus minimizing the chances of transferring microenvironmental elements. Marrow from young NZB donors reconstituted surface IgM-expressing B cells and CFU-B (B-cell colony-forming unit) in the marrow of SCID mouse recipients to levels comparable to that observed with donor NZB.xid marrow. The latter mice carry the xid gene that ameliorates the defects exhibited by B lineage cells of NZB mice. Both the number of surface IgM-expressing B cells and CFU-B were higher in the spleen of SCID mice that received NZB grafts than marrow cells from donor BALB/c or NZB.xid mice. Marrow from young NZB donors also reconstituted Thy-1, L3T4 and Lyt2-expressing cells in the spleen to levels higher than observed with young NZB.xid donor cells. The transplantation of marrow from 6-month-old NZB donors made it possible to test whether B lineage cells were present in that tissue and could mediate reconstitution in the normal SCID environment. Marrow from old NZB donors did reconstitute B cells in the marrow and spleen of SCID recipients. The level of reconstitution was comparable to that mediated by young BALB/c cells and twice that of old NZB.xid donor cells. The absolute number of splenic CFU-B was also higher in recipients of old NZB marrow as compared to young BALB/c cells. Old NZB.xid donor marrow reconstituted splenic Thy-1, L3T4 and Lyt2 T cells to levels less than observed with NZB donor cells. Analysis of serum Ig in recipients of old NZB cells indicated higher levels of total IgM as compared to mice engrafted with NZB.xid cells, and anti-single stranded DNA antibodies were detected.     

Altered selection processes of B lymphocytes in autoimmune NZB/W mice, despite intact central tolerance against DNA

Anti-DNA autoantibodies are the hallmark of systemic lupus erythematosus and the (NZBxNZW)F1 (NZB/W) murine model. To investigate potential defects in B cell tolerance, we followed the development of anti-DNA-specific B cells in 2-5-month-old mice transgenic for an unmutated muH chain in the normal C57BL/6 and in the NZB/W background. When the transgenic H chain was combined with a random kappa L chain repertoire about 60% of the antibodies bound to DNA. The analysis of the B cell repertoire in the spleen showed extensive receptor editing and a deletion of DNA reactivity in the C57BL/6 as well as in the autoimmune NZB/W background. NZB/W compared to C57BL/6 transgenic mice had a higher frequency of anti-DNA B cells among follicular B cells that were not censored by central tolerance mechanisms. Furthermore, positive selection of B cells with a recurrent rearrangement into the marginal zone compartment was more pronounced in NZB/W mice. Serum levels of transgenic IgM and of anti-DNA autoantibodies indicate a polyclonal activation of hyperactive B cells in the transgenic NZB/W mice. We propose different B cell receptor signaling thresholds for the NZB/W compared to C57BL/6 B cells. This could explain the quantitative differences in the B cell repertoire as well as the hyperactivity of B cells from NZB/W mice.     

Characterization of the spontaneous autoimmune (anti-erythrocyte) response in NZB mice using a pathogenic monoclonal autoantibody and its anti-idiotype.

A monoclonal anti-mouse RBC antibody (G-8) has been prepared that appears to represent a pathogenic autoantibody related to those that arise spontaneously in aging NZB mice and which cause autoimmune haemolytic disease (AIHD). When G-8-producing hybridoma cells were grown as tumours in BALB/c mice, the mice developed AIHD characterized by a decrease in the number of erythrocytes (haematocrit) and the development of Coombs-positivity. An anti-idiotypic antibody (E8) was prepared against G-8 and was found to recognize an idiotypic determinant present on most autoantibody-forming cells derived from old (Coombs-positive) NZB mice. The results suggest that the IgM autoantibody response of NZB mice to self-erythrocytes is restricted to a limited number of clones, most of which express the G-8 idiotype.     

Increased yields of IgG2a- and IgG3-secreting hybridomas after fusion of B cells from mice with autoimmune diseases

Hybridoma technology has made the production of antigen-specific monoclonal antibodies feasible and almost routine, but the production of certain biologically desirable antibody isotypes has remained difficult. Three strains of autoimmune mice (MRL/l, NZB, and BXSB) were compared to a normal strain (BALB/c), in fusions with a BALB/c myeloma (NS-1) in order to study the rescue of relevant isotypes with the desired antigenic specificities. Mice from these four strains were immunized with colon carcinoma cells, and the hybridoma supernatants from thirty fusions were analyzed for (1) reactivity with cell surface determinants on the immunizing cell line; and (2) Ig class and subclass isotypes. We found that compared to BALB/c mice, MRL/l mice produced greater numbers, and NZB and BXSB mice comparable numbers, of cell surface-reactive hybridoma clones per fusion. MRL/l mice produced the largest number and highest percentage of cell-surface reactive IgG2a (22.4%) and IgG3 (10.6%) producing clones, followed by NZB mice which produced predominantly IgG2a clones (12.3%). BXSB mice, which have latent autoimmune disease, showed no significant difference from normal BALB/c controls (IgG2a:0.7% and IgG3:1.9% vs. IgG2a:4.8% and IgG3:4.8%). The increase in IgG2a and IgG3 clones derived from MRL/l mice was age-dependent, correlating with the age at which abnormal proliferation of T cell and splenic enlargement occurs (2-4 months). We conclude that MRL/l mice are useful for generating monoclonal antibodies of the IgG2a or IgG3 isotype, provided fusions are performed at the time of maximal lymphoproliferation.     

Interleukin 2 is a proliferative signal for B cells from autoimmune mice

T cells from murine lupus strains manifest complex defects in interleukin 2 (IL 2) production and receptor expression. The capacity of B cells from such mice to utilize IL 2 as a growth factor has not been previously reported and is examined herein. Anti-Thy-1.2 plus complement-treated spleen cells from 6-8-week-old autoimmune MRL-lpr/lpr mice and from age and sex-matched immunologically normal CBA/J mice were cultured with lipopolysaccharide (LPS) for 36 h and analyzed for the expression of IL 2 receptors using the monoclonal antibody 7D4. The percentage of B cells expressing IL 2 receptors was comparable in MRL-lpr/lpr and CBA/J mice. In contrast to those from CBA/J, BALB/c and (BALB/c X NZW)F1 mice, LPS-stimulated B cells from MRL-lpr/lpr and from (NZB X NZW)F1 mice were capable of proliferating in response to IL 2. Fractionation of MRL-lpr/lpr B cells using Percoll gradient density separation demonstrated that the IL 2-responsive population consisted predominantly of large cells. In addition, unfractionated B cells from MRL-lpr/lpr mice were found to be substantially more responsive to IL 2 than those from CBA/J and BALB/c mice following activation with anti-immunoglobulin plus LPS. The hyper-responsiveness to IL 2 may be a consequence of the state of activation of autoimmune B cells and is of potential importance in the pathogenesis of systemic lupus erythematosus.     

Idiotypes of monoclonal anti-DNA antibodies produced in autoimmune B/W mice are expressed in normal mice.

An anti-idiotypic antiserum was prepared in a rabbit immunized against a pool of six monoclonal anti-DNA antibodies generated in B/W mice. This antiserum detected idiotypic determinants in four of the six monoclonal anti-DNA antibodies but also in the serum of several non autoimmune strains (BALB/c, NZB X BALB/c) F1 hybrids & CBA/LH). The antiserum also reacted, but only to a weak degree, with B/W mouse sera. These results indicate that some idiotypes of anti-DNA antibodies produced by autoimmune B/W mice are present in normal mouse sera.     

The role of suppressor T cells in the expression of autoimmune haemolytic anaemia in NZB mice.

The course of haemolytic anaemia in NZB mice has been altered by injection of spleen cells from diseased mice into younger ones before the onset of clinical disease. Recipients greater than 6 weeks of age developed early-onset autoimmune disease, recipients less than 6 weeks of age developed early-onset autoimmune disease, recipients less than 6 weeks of age recovered from early induced disease and showed a delay in the onset of spontaneous disease as compared with untreated NZB mice. This delay was due to the induction in the young mice of splenic suppressor cells. These cells were non-adherent to nylon wool and suppressed autoantibody formation on transfer to old Coombs-positive recipients. Suppressor cells active against autoantibody formation on transfer to old Coombs-positive recipients. Suppressor cells active against autoantibody-producing cells may be present in young untreated NZB mice, but not in sufficient numbers to suppress autoantibody production on adoptive transfer to Coombs-positive; however, when Ig-negative cells from the spleens of very young NZB mice were transferred together with Ig-positive cells from Coombs-positive donor mice to irradiated NZB recipients, the autoantibody production of the transferred B cells was suppressed in some cases. Suppressor cell activity could also be induced by co-culture of spleen cells from old Coombs-positive and young Coombs-negative NZB mice in vitro.     

Proliferative responsiveness of B cells from autoimmune NZB mice to anti-immunoglobulin and interleukin-4.

Proliferative responses of B cells to anti-IgM antibody and recombinant interleukin-4 (rIL-4) were studied in autoimmune NZB mice. While anti-IgM antibody and rIL-4 act synergistically on resting B cells to induce proliferation in BALB/c and C57BL/6 mice, they failed to produce synergistic effect on NZB B cells, although these NZB B cells responded better to the individual stimulus with anti-IgM antibody or rIL-4. Cell-fractionation analysis of NZB splenic B cells using Percoll density centrifugation showed marked increase in low-density B cells and decrease in high-density B cells. Proliferative response patterns of each subpopulation of NZB B cells were not different from those of control BALB/c B cells, except for higher response of NZB B cells to anti-IgM antibody. Thus, the accumulation of large low density B cells, probably resulting from in vivo activation, may partly account for the altered responsiveness of NZB B cells as a whole.     

Hybridoma autoantibodies to erythrocytes from NZB mice and the induction of hemolytic anemia

We established two clones of monoclonal hybridoma, from a non-immunized NZB mouse, which produce IgM class hemagglutinating autoantibodies reactive with the exposed murine erythrocyte autoantigens. Absorption studies revealed that one monoclonal antibody exhibits cross-reactivity to chick erythrocytes and mouse liver, and the other antibody to rat erythrocytes and mouse brain. Optimal temperatures for the hemagglutination were 22 degrees C with the former and 4 degrees C with the latter. The specificity and nature of the autoantibodies are apparently distinct from any of the erythrocyte autoantibodies described to date in NZB mice, anti-X, anti-HB, anti-HOL or anti-I antibodies. Implantation of these hybridoma cells in BALB/c mice induced autoimmune hemolytic anemia associated with a marked splenomegaly. These findings provide evidence that the erythrocyte autoantibodies in NZB mice are more heterogenous than generally assumed and suggest that varieties of erythrocyte autoantibodies may be involved in the development of a naturally occurring hemolytic disease in NZB mice.     

Abnormal behavior of gamma-committed B lymphocytes probed by a lymphocyte blastogenesis inhibitory factor in autoimmune MRL mice

Recently, we have characterized a lymphocyte blastogenesis inhibitory factor (LBIF) which was purified from the culture supernatant of a human histiocytic lymphoma U-937 (Sugimura, K. et al., Eur. J. Immunol. 1989. 19: 1357). In this study, we investigated the effect of LBIF on the antibody production of autoimmune MRL mice in vitro. We demonstrated here that (a) LBIF inhibited the IgM, IgG and IgA antibody responses of lipopolysaccharide (LPS)-stimulated spleen cells of normal BALB/c mice, (b) in the case of old autoimmune MRL/Mp-lpr/lpr (MRL/l) and MRL/Mp-(+)/+ mice, however, LBIF inhibited IgM and IgA but not IgG responses of LPS-stimulated spleen cells, (c) the antibody production of all IgG subclasses, IgG3, IgG1, IgG2b and IgG2a, was not sensitive to LBIF inhibitory activity in these autoimmune mice, (d) in young MRL mice (3-5-week-old MRL/l), which were phenotypically normal, LPS-induced antibody production of all isotypes (IgM, IgG and IgA) was strongly inhibited by LBIF as shown in normal BALB/c mice and (e) in the case of 7-week-old MRL/l the insensitivity to LBIF was concomitant with the appearance of gamma + B lymphocytes. Thus, by employing LBIF as a probe, this study showed a correlation between the pathogenesis of MRL autoimmune disease and the lack of LBIF sensitivity of hyperactive B lymphocytes and suggested that the intrinsic abnormality of autoimmune MRL B lymphocytes might be confined to gamma- but not mu- or alpha-committed B cells.     

Active role of T cells in promoting an in vitro autoantibody response to self erythrocytes in NZB mice.

The in vitro anti-self erythrocyte antibody response of NZB spleen cells appears to be influenced directly by T cells. Thy-1+, L3T4+ helper T cells are required for: (i) the generation in vitro of MRBC-specific IgM and IgG AFC by spleen cells from 'autoimmune' (9-12-month old) NZB mice and (ii) the generation in vitro of MRBC-specific IgM and IgG AFC by spleen cells depleted of suppressor cells from pre-autoimmune (2-3-months-old) NZB mice which show no clinical signs of an anti-MRBC response. It is evident from the present and previous studies that the anti-MRBC autoantibody response is regulated in pre-autoimmune spleen cell populations by Ly2+ T cells. Ly2-T cells from both pre-autoimmune and autoimmune mice in sufficient numbers can overcome this normal regulation and promote the anti-MRBC response in cultures of unfractionated pre-autoimmune spleen cells. Ly2- T cells isolated from autoimmune NZB mice were consistently more active in this than the Ly2- T cells isolated from pre-autoimmune mice, suggesting an enrichment of MRBC-reactive Ly2- T cells in autoimmune NZB mice. The Ly2- T cells from autoimmune NZB mice greatly enhance the autoimmune anti-MRBC response relative to a modest enhancement of the response to a foreign antigen, SRBC, produced by the same cells. These data indicate that T cells play an important role both in supporting the autoantibody response to MRBC and in disrupting tolerance, leading to autoimmunity in NZB mice.     

Autoreactive T cell specificity in autoimmune hemolytic anemia of the NZB mouse

Splenic T cells from Coombs'-positive New Zealand Black (NZB) mice proliferated consistently in vitro in response to the integral red blood cell (RBC) membrane protein Band 3, the antigen previously shown to be the target for the pathogenic RBC autoantibodies. The responding cells predominantly express CD4 and the proliferative response is blocked by antibodies to the NZB major histocompatibility complex class II but not by antibodies to an irrelevant H-2 haplotype. NZB splenic T cells also proliferated in response to the internal membrane skeleton protein spectrin. By contrast, T cells from BALB/c and DBA2 mice, which bear the same H-2 haplotype as NZB mice, but which do not develop autoimmune hemolytic anemia (AIHA), fail to respond to Band 3. It is considered that these results support the hypothesis that Band 3-reactive T cells provide help for the production of pathogenic anti-Band 3 autoantibodies in NZB mice. T cells from Coombs'-negative NZB mice as young as 3 weeks old proliferated in response to Band 3; moreover, the RBC from Coombs'-negative mice bore elevated levels of autoantibody as judged by a sensitive direct enzyme-linked anti-globulin test. Thus, the pathology of AIHA develops at a much earlier age than was thought previously.     

Resistant nature of T cells of autoimmune mice to tolerance induction with human serum albumin

Inducibility of immunological tolerance to a T-dependent antigen, human serum albumin, was studied in autoimmune mice (NZB, MRL/Mp-lpr/lpr and male BXSB mice), comparing with non-autoimmune mice (BALB/c, DBA/2, MRL/Mp-+/+ and female BXSB mice). Weekly injections of increasing doses of the tolerogen into DBA/2 mice induced T-cell tolerance without the participation of suppressor cells and without affecting B cells. All of the autoimmune mice tested were refractory to the tolerogen; partial tolerance (40%-60% responsiveness) was attained in mice aged 1 month, but not in older mice. By comparison, tolerance was induced in non-autoimmune mice to a similar degree irrespective of the age of the mice. The tolerance inducibility was also examined in reciprocal bone marrow cell (BMC) transfer experiments between NZB and DBA/2 mice and also BMC transfer into B10-D2 mice. It was shown that recipients of NZB BMC were more resistant to tolerance induction than those of DBA/2 BMC, while irradiated NZB mice receiving DBA/2 BMC were susceptible. Hence, it is suggested that the tolerance-resistant nature of the NZB mice was inherent in T-cell precursors in the bone marrow and not in the host environment. Similar findings in support of this notion were also observed in BSXB mice. The significance of tolerance resistance in the development of autoimmunity is discussed.     

Self-limited autoimmune disease related to transient donor B cell activation in mice neonatally injected with semi-allogeneic F1 cells.

BALB/c mice injected at birth with 10(8) semi-allogeneic (C57BL/6 x BALB.IgHb)F1 spleen cells develop a lupus-like syndrome in which autoantibodies bear exclusively the donor allotype. We have analyzed the evolution of donor B cell chimerism and the autoimmune manifestations during the first year of life in these mice. Anti-DNA, -histone, and -cardiolipin IgG antibodies as well as circulating immune complexes appeared in the second week of life, reached the highest values around the sixth week, and then progressively dropped to normal values after the sixth month in most mice. The kinetics of the evolution of the autoimmune manifestations, as well as the kinetics of serum donor Ig allotype, were parallel to the kinetics of donor B cell chimerism, which was particularly prominent in the spleens in early weeks of life, and progressively decreased after remission of the autoimmune syndrome. Membrane-proliferative glomerulonephritis, which was followed as the more representative histological abnormality in this model, was particularly evident after 10 weeks of life, but disappeared by the end of the follow-up. Interestingly, when mice with a self-limited disease were re-injected with 10(8) F1 spleen cells i.v., a flare in the serological manifestations was observed. In these re-injected mice a predominance of anti-DNA, IgG1 antibodies bearing exclusively the donor allotype was also observed, as in the early weeks of life.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphocyte subpopulations in the thymus of NZB x NZW mice: phenotypic characterization by flow cytofluorometry analysis.

The composition of the thymocyte population was investigated as a function of age in the autoimmunity-prone NZB x NZW F1 (NZB x W) female mice and in control BALB/c female mice. Single- and two-colour flow cytofluorometry analyses were used to quantitate the cell surface binding of fluorochrome-conjugated antibodies directed against various lymphocyte markers and of fluoresceinated peanut agglutinin (PNA). In both mouse strains, two major phenotypically distinct thymocyte subpopulations were thus identified. The predominant subpopulation was characterized as bright Thy-1+, Lyt-1 + 2+ and bright PNA+, and the other one as dull Thy-1+, Lyt-1 + 2- and dull PNA+. The relative frequencies of these two subpopulations were similar in NZB x W and BALB/c mice at 3 months of age. However, from 6 months onwards, slight but significant differences became detectable between the two strains. Thus, in BALB/c mice, both thymocyte subpopulations regressed at approximately the same rate during ageing so that their relative proportions remained constant. In contrast, in NZB x W mice, while the number of bright Thy-1+ cells diminished as in BALB/c mice, the number of dull Thy-1+ cells barely varied from 3 to 12 months of age, which resulted in a proportional increase of this latter subpopulation. Moreover, elevated frequencies of surface immunoglobulin-bearing cells were recorded in the thymus of 8-12 month old NZB x W mice but not in BALB/c mice. Therefore, the development of autoimmunity in NZB x W mice appears associated with an abnormal age-dependent evolution of the intrathymic lymphocyte population.     

Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells

Our recent studies on an autoantibody-transgenic mouse linedemonstrated that peritoneal B-1 cells are responsible for autoimmunesymptoms. However, whether B-1 cells in the peritoneum are generallyinvolved in the pathogenesis of autoimmune disease remains controversial.To test the possible involvement of peritoneal B-1 cells inautoimmune symptoms of autoimmune-prone NZB mice, we eliminatedthe peritoneal cells by hypotonic shock with repeated I.p. injectionof distilled water every 7 days into neonatal or 8-week-oldNZB mice. By this treatment, B-1 cells, which self- renew withinthe peritoneal cavity, are expected to be preferentially eliminated,while other peritoneal cells can be easily supplied from bonemarrows after this treatment indeed, in distilled water-treatedold NZB mice, the number of B-1 cells decreased in spleen aswell as in lamina propria of the gut but the numbers of conventionalB cells and T cells did not change. Moreover, the productionof autoantibodies against erythrocytes significantly decreasedand the occurrence of autoimmune hemolytic anemia was reducedin 12-month-old treated NZB mice. Similarly, the eliminationof peritoneal cells of NZB/NZW (NZB/W) F₁; mice by water injectiondecreased anti-DNA IgG antibodies in the sera and reduced thepathological changes of the kidney. These results suggest thatperitoneal B-1 cells may be a source of autoantibody-producingcells in autoimmune diseases of NZB and NZB/W F₁; mice.     

Immunologic abnormality in NZB/W F1 mice. Thymus-independent expansion of B cells responding to interleukin-6.

We have previously reported that B cell abnormality in NZB/W F1 mice developed independently of thymus. Here we examined further whether B cells from NZB/W F1 mice respond to interleukin-6 (IL-6), a factor for terminal differentiation of B cells. When freshly isolated splenic B cells were incubated for 5 days in the presence of human IL-6, an increased production of both IgM and IgG, including anti-DNA antibody, was evident in NZB/W F1 mice; there was no increase in BALB/c mice. A magnitude of augmentation in IgG but not IgM production by IL-6 became more apparent in older NZB/W F1 mice. The increased immunoglobulin production seen with IL-6 was neutralized by treatment with rabbit anti-recombinant human IL-6 antibody. As B cells from athymic NZB/W F1 nude mice also responded to IL-6, it was suggested that B cells in NZB/W F1 mice differentiated into the IL-6-responding state in a thymus-independent manner. This B cell abnormality may be associated with the pathogenesis of autoimmune disease in NZB/W F1 mice.