Bromelain-treated Mouse Erythrocytes Research Articles

Mycobacteria-induced autoantibody production is associated with susceptibility to infection but not with host propensity to develop autoimmune disease.

Mycobacteria cause increase in autoantibody production in the host during the first weeks of infection. The level of the autoantibody enhancement varies widely in different hosts, suggesting that it depends on features of the host make-up. We have investigated the participation of two characteristics of the host in the modulation of mycobacteria-induced autoantibody production: (i) the host being genetically determined to later develop spontaneous autoimmune disease; (ii) the host being susceptible/resistant to mycobacterial infection. Mycobacterium avium infection was studied in 3-month-old mice that are prone (NZB and C57B1/6-lpr/lpr strains) or not (NZW and C.D2 strains) to develop, when older, autoimmune disease; these murine strains are either naturally susceptible (C57B1/6-lpr/lpr and NZW) or resistant (NZB and C.D2) to mycobacteria. Mycobacterium avium infection was produced by i.p. injection of 3 x 10(7) viable bacilli. At days 15 and 30 of the infection, we determined the following parameters: (i) number of cells producing natural autoantibodies (splenic cells showing surface antibodies against bromelain-treated mouse (BrM) erythrocytes); (ii) suppression of the primary response to T cell-dependent antigen (i.e. to sheep erythrocytes); (iii) immunoglobulin classes and IgG isotypes; (iv) titres of anti-dsDNA antibodies; and (v) serum concentrations of interferon-gamma (IFN-gamma). We found that the highest elevations in natural autoantibodies were associated with hosts being naturally susceptible to mycobacteria, but not with the host being genetically determined to later develop autoimmune disease. The rise in autoantibodies was predominantly of the IgM type, being associated with suppression of the T cell response and accompanied by increase in serum IFN-gamma. Mycobacteria failed to induce any significant enhancement in pathogenic anti-dsDNA antibodies. Our data suggest that the finding of a high level of autoantibodies during the early phase of mycobacterial infection reflects host susceptibility of the infectious agent, and that it is not related with its propensity to later develop autoimmune disorders.

Frequent occurrence of identical heavy and light chain Ig rearrangements.

Single-cell PCR analyses of expressed Ig H and L chain sequences presented here show that certain rearrangements occur repeatedly and account for a major segment of the well-studied repertoire of B-1 cell autoantibodies that mediate the lysis of bromelain-treated mouse erythrocytes, i.e. antibodies reactive with phosphatldyicholine (PtC). We repeatedly isolated at least 10 different types of VH region rearrangements, involving three distinct germline genes, among FACS-sorted PtC-binding B-1 cells from three strains of mice (C57BL/6J, BALB/c and C.B-17). The predominant rearrangement, VH11-DSP-JH1 (VH11 type 1), has been previously found in anti-PtC hybridomas in several studies. We show that within each of six mice from two strains (C57BL/6J and BALB/c), unique instances of IgH/IgL pairing arose either from different B cell progenitors prior to IgH rearrangement or from pre-B cells which expanded after IgH rearrangement but prior to IgL rearrangement. Together with other recurrent rearrangements described here, our findings demonstrate that clonal expansion of mature B cells cannot account for all repeated rearrangements. As suggested by initial studies of dominant idiotype expression, these findings confirm that clonal expansion is only one of the mechanisms contributing to the establishment of recurrent rearrangements.

Quantification of natural antibody producing B cells in rats by an improved ELISPOT technique using the polyvinylidene difluoride membrane as the solid support

We describe here a new type of solid support for the ELISPOT assay, the PVDF membrane. In parallel tests, spot yields on this membrane were superior to those obtained with the frequently used nitrocellulose (NC) membrane, coated with the same rat anti-IgM and anti-IgG antibodies, incubated with the same rat spleen cell suspensions, and developed with the same combination of AP-labeled conjugates and substrate. We therefore used the PVDF membrane, coated with anti-rat IgM and IgG antibodies, ssDNA or bromelain-treated mouse erythrocytes (BrMRBC) (exposing phosphatidylcholine (PC) as major autoantigen) to develop ELISPOT assays for the quantification of isotype-specific natural antibody secreting cells (ASC) in rats. We confirmed the isotype specificity of the binding of the anti-rat IgM and anti-rat IgG coating antibodies and conjugates with the secreted rat antibodies in this assay, and, by inhibition of spot formation with soluble antigen, their specificity for ssDNA and BrMRBC. An in-house 18-well culture device for the easy manufacture of PVDF-lined culture wells greatly facilitated coating, blocking, and washing procedures, as compared to the original method in 24 well culture plates. This simple, fast, specific and sensitive ELISPOT assay was used to make an inventory of the numbers of natural splenic ASC in Wistar and Fischer rats.

Autoimmune Effects of Hexachlorobenzene in the Rat

We investigated whether autoimmune disregulation underlies the formerly reported induction of IgM hypergammaglobulinemia and lymphoid organ enlargement by hexachlorobenzene (HCB) in rats. To this end blood, liver, and lymphoid organs were collected from male Wistar rats after feeding a semisynthetic diet containing 0, 500, or 1000 mg HCB/kg for 3 weeks. Sera prepared from the blood were analyzed for total and (auto)antigen-specific antibody levels by ELISA, organs were weighed, and spleens were further investigated morphogically using immunohistochemically stained cryosections. Present experiments confirmed the ability of HCB to increase total IgM, but not IgG, levels and to increase relative spleen, lymph node, and liver weights. HCB treatment elevated IgM, but not IgG, levels against single-stranded DNA, native DNA, rat IgG (representing rheumatoid factor), and bromelain-treated mouse erythrocytes that expose phosphatidylcholine as a major autoantigen. Antibody levels against the foreign antigens sheep erythrocytes, tetanus toxoid, and bovine serum albumin remained unaffected. The IgM autoantibodies proved to be polyreactive. Morphometric analysis of spleen sections showed that HCB caused a proportionally equal expansion of all splenic compartments, but when individual spleen weights were taken into account a significantly larger expansion of the predominantly B cell-containing marginal zones could be noted. The latter compartment also contained an increased number of macrophages that can be recognized by the monoclonal antibody ED3. The ability of HCB to elevate serum antibody levels against autoantigens, but not foreign antigens, indicates that HCB probably does not act by polyclonal B cell activation. The IgM isotype, the repertoire, and the polyreactivity of the serum autoantibodies suggest that HCB activates a recently described B cell subset shown to be committed to the production of these autoantibodies and associated with various systemic autoimmune diseases. Since the marginal zone is considered to be the splenic lodging site of this B cell subset and since increases of ED3 + macrophages have been associated with autoimmune diseases in the rat, the observed changes of the marginal zones in HCB-treated rats is in line with this notion.

Effect of age on the expressed B cell repertoire: role of B cell subsets.

Aged humans and experimental animals are impaired in their responses to most foreign antigens although they produce greater amounts of autoantibodies. We have examined the effect of age on the production of antibodies to a prototypic foreign antigen, sheep erythrocytes (SRBC), and to a prototypic autoantigen, bromelain-treated mouse erythrocytes (BrMRBC), in young and old mice before and after immunization with SRBC. Old mice express more anti-BrMRBC plaque-forming cell (PFC) antibodies before and an even greater number after immunization with SRBC than young mice. Conversely, old mice produce far fewer anti-SRBC PFC than young mice following immunization with SRBC. We hypothesized that the differences in the responses of old mice to BrMRBC and SRBC reflects differences in the activity of CD5+ and CD5- B cells. To test this hypothesis we immunized young and old mice with foreign antigens reported (and confirmed in our studies) to stimulate CD5+ B cells [TNP-ficoll and phosphorylcholine-keyhole limpet hemocyanin (KLH)] or CD5- B cells (SRBC and TNP-KLH). We found that the PFC response of old mice to antigens mediated by CD5+ B cells was equal to or greater than that of young mice. In contrast the PFC response of old mice induced by antigens mediated by CD5- B cells was only 10% that of young mice. Thus it appears that the immune response of old mice is well maintained for antigens which elicit a CD5+ B cell response but not for those which elicit a CD5- B cell response.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies to phosphatidylcholine. The murine antibromelain RBC response.

The observation that murine B-cell populations can contain relatively large numbers of cells that produce IgM with the ability to lyse bromelain-treated mouse erythrocytes (BrMRBC), but not normal untreated MRBC, was made nearly 20 years ago. The major observations regarding the antigen specificity, the cells that produce this IgM, and the immunoglobulin V genes that encode them are summarized in this report. The epitope on BrMRBC that is recognized has been identified as the head group of phosphatidylcholine (PtC); B cells whose IgM has this specificity can be easily identified by their ability to bind fluorescent synthetic liposomes whose membrane contains PtC. The cells producing IgM specific for PtC all derive from the Ly-1 B-cell subset, and they use primarily two VH/VL gene pairs to encode the anti-PtC antibodies. The VH genes used describe two new VH gene families, VH11 and VH12. The genes encoding anti-PtC are unmutated and have characteristics and restricted VDJ constructions. The cells with this specificity, within individual mice, are polyclonal. These criteria are consistent with a primary antigen-driven clonal selection mechanism as the basis for the development of this immune specificity.

Properties of murine antibodies from different V region families specific for bromelain-treated mouse erythrocytes.

We have analyzed the combining site diversity of murine antibodies reactive with bromelain-treated mouse RBC (BrMRBC) in B10.A mice. Although several VH and V kappa genes are used to generate antibodies of this specificity, two combinations account for more than 80% of the repertoire from the spleen: VH11/V kappa 9 and VH12/V kappa 4. Antibodies representing these two predominant groups were found to correspond to two previously reported distinct cross-reactive Id families accounting for most of the anti-BrMRBC reactivity in several strains of mice. mAb of the VH11/V kappa 9 type bound the haptens trimethyl-ammonium and phosphorylcholine much more avidly (approximately 1000 fold) than did the VH12/V kappa 4 type antibodies. Both of these haptens represent constituents of phosphatidylcholine, which BrMRBC-specific antibodies are reported to bind. Despite this differential reactivity, members of both the VH11/V kappa 9 and VH12/V kappa 4 antibody groups lysed BrMRBC with similar efficiencies. These data suggest that the two major classes of BrMRBC-specific antibodies have at least partially different specificities and imply that the events that lead to their high frequencies in the CD5+ repertoire may result from different selective forces.

Influence of the oral administration of excess copper on the immune response

We have studied the influence of the oral administration of excess copper (Cu) on the immune response. With this aim, mice maintained on standard laboratory diet received 50, 100, 200, or 300 ppm of Cu as copper sulfate in the drinking water during 3 to 10 weeks. Inhibition of the proliferative response to concanavalin A was observed in mice exposed to 100 ppm of Cu for 8 weeks and to 200 ppm of Cu for either 3 or 8 weeks. Conversely, a significant increase in the proliferative response to Escherichia coli lipopolysaccharide (LPS) was observed in mice exposed to 50 or 100 ppm of Cu for 3 weeks. However, the response to LPS was also significantly inhibited following prolonged Cu administration. In contrast, mice exposed to low or high Cu doses during short or long periods showed increased production of autoantibodies directed to bromelain-treated mouse erythrocytes. The DTH response to sheep red blood cells was not modified following short-term administration of 100 ppm of Cu, but was depressed after prolonged exposure to this dose of the metal. Significant inhibition of the DTH response was observed in mice exposed to 300 ppm of Cu for 5 or 10 weeks. Thus, oral administration of excess Cu altered the immune response in a fashion related to the dose and duration of treatment.

Antiphospholipid Antibodies Reactive with Bromelain-Treated Mouse Erythrocytes in Mice, Rats and Rabbits

It has been reported that normal mice have a large number of B cells against bromelain-treated mouse erythrocytes (BrMRBC) and the anti-BrMRBC antibodies are reactive with liposomes of phosphatidylcholine and a phospholipid moiety of low-density lipoproteins from chicken egg yolk (EyLDL). In this study, we detected anti-BrMRBC hemolytic antibodies in the normal sera of rats and rabbits as well as mice. The antibodies in all the three species of animals bound to the columns of EyLDL. The EyLDL-binding antibodies could react with both liposomes of phosphatidylcholine and BrMRBC. The immunization with EyLDL evoked a vast production of anti-BrMRBC antibodies in rats and rabbits as well as mice. The anti-BrMRBC antibodies in the immunized animals could also react with liposomes of phosphatidylcholine. These findings suggest that rats and rabbits have the equivalent of mouse anti-BrMRBC B cells.

Binding of low-density lipoproteins from chicken egg yolk to mouse anti-phospholipid B cells which include B cells against bromelain-treated mouse erythrocytes

Low-density lipoproteins from chicken egg yolk (EyLDL), which are reactive with mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC), were conjugated with fluorescein isothiocyanate (FITC). FITC-EyLDL could bind specifically to mouse anti-phospholipid B cells, which comprised all the BrMRBC-rosette-forming cells and anti-BrMRBC lipopolysaccharide-reactive B cells, C3H/He mice at 12 weeks of age had, approximately, 7 × 10 5 EyLDL-binding cells in the peritoneal cavity, 3 × 10 5 EyLDL-binding cells in the pleural cavity, and 3 × 10 5 EyLDL-binding cells in the spleen. In ontogeny, the numbers of EyLDL-binding cells in the peritoneal cavity expanded greatly by 4 weeks. Other normal strains of mice and C3H/HeJ mice at 12 weeks of age had 4–7 × 10 5 EyLDL-binding cells in the peritoneal cavity; the numbers were large (19 × 10 5) in NZB mice, rather small (2 × 10 5) in MRL/ lpr mice, and very small (0.1 × 10 5) in CBA/N mice. In some of various strains of mice at 12 months of age, more than 20% of peritoneal cells were EyLDL-binding cells; in particular, all of five older NZB mice examined had more than 10 7 EyLDL-binding cells in the peritoneal cavity.

Interleukin 5 Regulation of Peritoneal B‐Cell Proliferation and Antibody Secretion

The influence of recombinant interleukin 5 (rIL-5) on murine peritoneal B-cell proliferation and antibody secretion was examined. Larger, low buoyant density peritoneal B cells proliferated better with rIL-5 than the smaller resting B cells. this was also true for splenic B cells; however, comparison of the respective populations showed the large peritoneal B-cell responses to be superior. Limiting dilution analyses showed that from 25% to about 40% of large peritoneal B cells proliferated in response to rIL-5 when lipopolysaccharide (LPS) was present. No detectable difference in the fraction of proliferating splenic B cells was seen in the presence of rIL-5. These results are consistent with expression of IL-5 receptors on about 70% of low-density peritoneal B cells as determined by fluorescent staining with anti-Il-5 receptor monoclonal antibody (MoAb). IL-5 also enhanced spontaneous and mitogen-driven IgM secretion by both peritoneal and splenic B lymphocytes; the increases exhibited by peritoneal B cells, however, were at least twice those exhibited by splenic B cells. Spontaneous and mitogen-driven secretion of auto-antibodies to bromelain-treated mouse erythrocytes (BrMRBC) by peritoneal B cells were also increased by this interleukin. Furthermore, rIL-5 enhanced peritoneal B-cell plaque-forming cell (PFC) responses to TNP-LPS but not to TNP-Ficoll. Both an anti-IL-5R MoAb and an anti-IL-5 MoAb blocked the rIL-5-induced enhancement of proliferation and auto-antibody PFC responses. Hence, IL-5 appears to be important for the regulation of proliferation and antibody secretion by many murine peritoneal B cells.

Estrogen induces the development of autoantibodies and promotes salivary gland lymphoid infiltrates in normal mice

There are important bidirectional interactions between the immune and the endocrine system. Sex hormones influence the immune system throughout life including postnatal and prenatal stages. For example, we find that administration of estrogen to normal mice markedly augments the ability of CD5 + B cells to express their autoimmune potential by producing increased numbers of plaque-forming cells (APFC) to bromelaintreated mouse erythrocytes (Br-ME) [1]. The effect of sex hormones on immune function at the most critical stage of development, the prenatal period, remains unstudied. We hypothesize that an imbalance of the in utero sex hormone microenvironment critically influences the fetal immune system. We have termed this influence immunological imprinting [2]. After birth this imprinting could contribute to immune-mediated disorders. To test this hypothesis, we developed a mouse model in which normal mice were prenatally exposed to estrogens. In preliminary experiments, these mice produced higher numbers of APFC to Br-ME, particularly in the peritoneal cavity cell exudates. Furthermore, mice prenatally exposed to estrogens had accelerated development of autoimmune salivary gland lesions indistinguishable from Sjögren's syndrome (SS) in humans. Further experiments are warranted to confirm these findings. The prenatal effects of estrogen may have relevance for familial and neonatal autoimmune syndromes.

Biased immunoglobulin variable region gene expression by Ly‐1 B cells due to clonal selection

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.

Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline.

5-15% of lymphocytes in the peritoneums of normal adult B10.H-2aH-4bp/Wts (2a4b) mice are CD5+ (Ly-1) B cells that recognize phosphatidyl choline (PtC), a phospholipid component of all mammalian cells. We produced a set of IgM-secreting hybridomas from the peritoneal cells of normal, adult 2a4b mice. We found that this set of hybridomas shows a similarly high frequency of antibodies specific for PtC (21 of 86) that also react with bromelain-treated mouse erythrocytes. Restriction fragment analysis of Ig gene rearrangements and analysis of expressed Ig idiotypes reveal that these cells use a restricted set of variable region genes to generate the PtC-specific antibodies. The Ig genes used by the PtC-specific hybridomas appear to be the same as those found in the PtC-specific Ly-1 B cell lymphomas, CH27 and CH34.

Lipopolysaccharide-reactive B cells against bromelain-treated mouse erythrocytes: preferential migration to and survival in the peritoneal cavity.

The ratio of lipopolysaccharide (LPS)-reactive B cells specific for bromelain-treated mouse erythrocytes (BrMRBC) to general LPS-reactive B cells is far higher in the peritoneal cavity of normal mice than in their spleen. To investigate the high concentration of anti-BrMRBC LPS-reactive B cells in the peritoneal cavity, spleen and peritoneal cells from (CBA/N X C3H/He)F1 female normal mice were injected intravenously and intraperitoneally into F1 male X-linked immunodeficient mice. Both groups of B cells in the intravenously transferred cell population were able equally to home in the spleen, but only anti-BrMRBC LPS-reactive B cells could be detected migrating into the peritoneal cavity. About half the anti-BrMRBC LPS-reactive B cells in the intraperitoneally transferred cell population could be recovered from the peritoneal cavity, but general LPS-reactive B cells were eliminated rather rapidly from the peritoneal cavity. Neither group of B cells could be detected migrating from the peritoneal cavity into the spleen. These findings suggest that the high concentration of anti-BrMRBC LPS-reactive B cells in the peritoneal cavity may be caused by their preferential ability to penetrate into the peritoneal cavity through circulation and survive there.

Induction of antibody response to bromelain-treated mouse erythrocytes by low-density lipoproteins from chicken egg yolk

The administration of low-density lipoproteins from chicken egg yolk (EyLDL) to mice induced T-dependent antibodies against bromelain-treated mouse erythrocytes (BrMRBC). The anti-BrMRBC antibodies produced antigen specifically by EyLDL appear to be identical to the antibodies produced polyclonally by lipopolysaccharide (LPS); both antibodies reacted with the liposomes of phosphatidylcholine and showed identical idiotypic patterns. It is suggested that the anti-BrMRBC antibodies may be produced antigen specifically by exogenous antigens to be useful to the mouse. The activation of anti-BrMRBC B cells specifically by EyLDL or polyclonally by LPS resulted in neither expansion nor regression of anti-BrMRBC LPS-reactive B cells. This finding indicates that the population size of anti-BrMRBC LPS-reactive B cells is controlled independently by exogenous specific or polyclonal stimuli.

Further studies on the determinant recognized by naturally-occurring murine autoantibodies reacting with bromelain-treated erythrocytes

Radioimmunoassays (RIA) have confirmed previous studies that trimethylammonium (TMA) or its derivatives constitute part of the determinant recognized by naturally-occurring antibodies (NOA) with the ability to interact with bromelain-treated mouse erythrocytes (BrMRBC). Further studies on this determinant revealed its presence on erythrocytes from several species in addition to mice. In most cases (except in chickens) the determinant was cryptic and could be exposed only after proteolytic treatment of the erythrocytes. The determinant was also found on certain murine lymphoma cells. We also found that bromelain was not the only enzyme that could be used to expose the determinant. Papain, but not trypsin, was able to unmask the determinant on mouse erythrocytes. Rabbit antibodies directed against the idiotypes of four different monoclonal BrMRBC-binding NOA were prepared. Direct RIA assays and inhibition assays showed that the different monoclonal BrMRBC-binding NOA shared a common idiotype specific to such antibodies. The common idiotype was detected in the serum from several mouse strains and in wild mice.

Reactivity of Mouse Antibodies against Bromelain-Treated Mouse Erythrocytes with Various Mouse Cells before and after Protease Treatment

A mouse monoclonal antibody against bromelain-treated mouse erythrocytes (BrMRBC) was conjugated with fluorescein isothiocyanate. Various cells from the blood and lymphoid tissues of mice were stained before and after protease treatment with the fluorescent antibody. Without protease treatment, only the platelets were specifically, though dully, fluorescent. Protease treatment made all the erythrocytes, the majority of platelets, thymus and bone marrow cells and a small part of the spleen cells brightly fluorescent. The reactivity of the antibody with cells depended on the temperature, being stronger at 0 than at 37 degrees C. The present findings demonstrate that various mouse cells besides erythrocytes bear the epitopes for anti-BrMRBC antibodies in exposed or hidden form.

Ontogenetic development of lipopolysaccharide-reactive B cells against bromelain-treated mouse erythrocytes in mouse lymphoid tissues

In lymphoid tissues of mice, there exist LPS-reactive B cells which can differentiate to IgM-secreting plaque-forming cells (IgM-PFC) and PFC secreting antibodies against bromelain-treated mouse erythrocytes (BrMRBC) by LPS activation. In this study, four groups of LPS-reactive B cells in spleen, peritoneal cavity and mesenteric lymph nodes from 2- and 10-week-old mice were compared and enumerated as precursors of IgM-PFC and anti-BrMRBC PFC on days 1 and 2 after LPS activation in quantitative culture conditions. The induction of each of four PFC responses in peritoneal cells was sensitive to LPS and anti-mouse IgM antibodies as much as the induction of the respective PFC response in spleen cells. The ratios of four groups of PFC to each other were different among three tissues and between two ages. These findings support the view that the four groups of LPS-reactive B cells in each tissue are mostly in distinct subpopulations of B cells from each other, and the respective groups of different lymphoid tissues at different ages belong to the same subpopulation.

Ontogenetic development of mouse B cells against bromelain-treated mouse erythrocytes: Idiotypic analysis

Ontogenetic development of mouse B cells against bromelain-treated mouse erythrocytes: Idiotypic analysis