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K O Cox

Publications and source records attributed to K O Cox.

At least 19 recordsLinked to original sources
PubMed1991

IgM natural autoantibodies against bromelain-treated mouse red blood cells recognise carbonic anhydrase.

Carbonic anhydrase (CA) from mouse erythrocyte membranes is recognised as an autoantigen in Western blotting experiments with FUB 1, a murine IgM monoclonal antibody that binds both phosphatidylcholine and bromelain-treated mouse red blood cells (BrMRBC). Serum from mice stimulated with lipopolysaccharide (LPS-serum) also recognises CA. From SDS-PAGE, and blotting experiments with whole mouse erythrocytes, we found two closely spaced glycoprotein bands in the 30 kD region that reacted with both FUB 1 and LPS-serum. One of the molecular weight markers, bovine carbonic anhydrase which is of a molecular weight of about 30 kD, electrophoresed in the same 30 kD region also reacted with these antibodies. Carbonic anhydrases from a range of mammalian species were found to be crossreactive with FUB 1 and LPS-serum by Western blotting, whereas human glycophorin A and human asialoglycophorin were not recognised by the antibodies. FUB 1 specifically recognises both native and denatured bovine carbonic anhydrase in ELISA assays. The serological identity of the determinants of CA and BrMRBC was confirmed by specific absorption of both FUB 1 and LPS-serum with BrMRBC and normal mouse erythrocytes. We propose that a native autoantigenic epitope on erythrocytes may be revealed by the proteolytic action of bromelain and that this determinant is associated, at least in part, with carbonic anhydrase.

Animals↗
PubMed1990

Mouse autoantibodies bind to a phospholipase-C-sensitive structure on red blood cells.

After culturing mouse peritoneal cells in vitro for 4 days, high numbers of cells can be detected that secrete autoantibodies against isologous red blood cells (RBC), modified with the proteolytic enzyme bromelain (Brom). Plaque-forming cell numbers against mouse Brom RBC were significantly reduced by pretreating mouse Brom RBC prior to haemolytic assay with phospholipase C, an enzyme that hydrolyzes phospholipids, notably phosphatidylcholine. In contrast, further treatment of mouse Brom RBC with Brom, neuraminidase, beta-chymotrypsin, trypsin, or papain had no effect on plaque-forming cell numbers. These results show that phosphatidylcholine is an integral part of the mouse RBC autoantigen exposed by Brom treatment.

Animals↗
PubMed1989

Expression in transgenic mice of class I histocompatibility antigens controlled by the metallothionein promoter.

To study the effects of increased expression of major histocompatibility complex class I molecules on the development of self-tolerance, transgenic mice were produced that expressed the H-2Kb gene under the control of the metallothionein promoter. Administration of zinc enhanced transgene expression in liver, kidney and exocrine pancreas. No evidence suggestive of an autoimmune response was found in transgene-expressing tissues in mice otherwise allogeneic to H-2Kb. Despite this lack of responsiveness in vivo, T cells could be stimulated in vitro to lyse H-2Kb-bearing target cells. No infiltration was detected in transgenic mice after irradiation and reconstitution with bone marrow cells. When spleen cells were used for reconstitution, however, dense lymphocytic infiltration was seen, particularly in the portal tracts of the liver, and this was accompanied by piecemeal necrosis and apoptosis of periportal hepatocytes. This aggressive response progressively diminished with time, and by 12 weeks after reconstitution many of the portal tracts were free of infiltration while the others showed no accompanying necrosis. The picture at this stage was similar to that seen in chronic persistent hepatitis. These results suggest that, in addition to negative selection in the thymus, peripheral mechanisms not involving clonal deletion or permanent clonal anergy can prevent immune responses to self molecules.

Animals↗
PubMed1989

Expression of class I histocompatibility antigens in defined tissues: effects on T cell function.

We have generated transgenic mice by introducing into fertilized eggs the major histocompatibility complex class I gene, H-2Kb, linked to various promoters that target gene expression to particular tissues. In one system the gene was expressed in the medullary epithelial cells of the thymus; these were unable to impose tolerance on differentiating T lymphocytes. These findings, together with those of other workers who showed that epithelial thymus grafts can impose tolerance, provide clear evidence of functional heterogeneity among thymus epithelial cells. In other transgenic models the gene was expressed in nonlymphoid tissues, such as the pancreatic islet beta cells or the hepatocytes, exocrine pancreas and kidney tubules. In all these cases the tissues were not subjected to autoimmune attack and the animals were specifically tolerant of H-2Kb-bearing cells in vivo, although some could generate cytotoxic T lymphocytes in vitro. Hence, although the intrathymic environment may be the dominant site for negative selection, it is clear that ancillary mechanisms exist in the periphery to ensure that tolerance is achieved to antigens not synthesized in the thymus.

Animals↗
PubMed1987

B-cells purified using azide give different responses in vitro to B-cells purified without azide.

Some established techniques for B-cell enrichment include azide in the media. Here, B-cells were enriched using a technique which transiently exposed the cells of 15 mM sodium azide. After 3-4 days in culture, the precursor frequency of autoantibody-secreting cells in this population was 0.113%. In contrast, when B-cells were enriched using a technique which eliminated their exposure to sodium azide, the number of autoantibody-secreting precursor cells detected in cultures was increased up to 20-fold. Therefore, it is concluded that transient exposure to azide affects functional activities of B-cells in vitro.

Animals↗
PubMed1987

Effect of whole-body irradiation on precursor frequency of autoantibody-secreting cells and plaque-forming cells specific for bromelain-modified erythrocytes.

Murine spleen cells and peritoneal cells were assayed for autoantibody production against bromelain-modified mouse erythrocytes (mouse brom-RBC) after whole-body irradiation at a dose of 5 Gy. Spleens were taken directly from mice 3 days after irradiation, or cultured for 3 days 24 h after irradiation, and assayed. Cells taken directly from irradiated mice had a higher number of plaque-forming cells (PFC) against mouse brom-RBC, compared with unirradiated mice, when the data were expressed as PFC/10(6) viable cells. This apparent increase can be accounted for by the reduced number of cells in the spleens from irradiated mice. In both spleen cells and peritoneal cells cultured in high cell densities, no difference was observed in the number of PFC in cells from irradiated or unirradiated mice. The detected precursor frequency of autoimmune B cells in both cell populations cultured in limiting dilution was found to be lower in cells from irradiated mice. In contrast to other published data, it was concluded from these investigations that whole-body irradiation, at a dose of 5 Gy, does not augment autoantibody production against mouse brom-RBC. It is proposed that the reason for this is the presence of a regulatory cell that is relatively radioresistant and, hence, not removed after whole-body irradiation at a dose of 5 Gy.

Animals↗
PubMed1987

T cells or their products are required for the differentiation of precursor B cells into antibody-secreting cells specific for a supposed T-independent self-antigen.

From our experiments and those of others in which cells were cultured at a density of 100,000 cells per well, it has been suggested that autoantibody production against mouse bromelain-treated erythrocytes (mouse brom-RBC) was independent of T cells, and further, was enhanced by the removal of T cells from responsive cell populations. Here it is shown in limiting dilution cultures that the autoimmune response is highly dependent on T cells or their products. B cells purified from the peritoneal cavities of untreated mice did not differentiate in vitro into autoantibody-secreting cells unless provided with signals from at least one of two types of accessory cells. These were plastic adherent cells and T cells, derived either from the peritoneal cavity or from established cell lines. Here it is shown that peritoneal T cells or T cells from the LBRM-33 cell line stimulated the differentiation of purified B cells in vitro in the absence of added mitogens. The accessory cell effect could be transferred in supernatants derived from T-cell cultures but not filler-cell cultures. Recombinant interleukin-2 (rIL-2) added to culture medium did not stimulate B cells directly, but could increase precursor frequencies when added to unfractionated peritoneal cell cultures, or B-cell cultures to which cells from a T-cell line had been added. From these results, it is concluded that the differentiation of precommitted peritoneal B cells in vitro into autoantibody secretors is at least partially dependent on T cells or lymphokines derived from them. Therefore, any proposed mechanisms for regulation of this autoimmune response should encompass the requirement for T cells or their products in the final differentiation stages to autoantibody secretion.

Animals↗
PubMed1986

Dependency of B cells on the presence of adherent cells, or factors derived from them, for the production of autoantibodies in vitro in the absence of cell division.

Peritoneal cells from untreated mice secrete autoantibodies after 3-4 days of in vitro culture, although the cells do not divide. Here, peritoneal cells enriched for B cells to contain 95% surface Ig-bearing cells, did not secrete autoantibodies after 3 days of in vitro culture unless plastic-adherent cells derived from the peritoneal cavity were cultured with the B cells. Cell-free media, taken from peritoneal adherent cells that had been cultured for 3 days in vitro, when added a final concentration of 50% in fresh culture medium to purified B cells, substituted for the presence of accessory cells. In contrast to cultures of unfractionated peritoneal cells, little increase in precursor frequency was detected when enriched B cells were cultured in the presence of LPS/DXS. However, the addition of adherent cells, supernatants derived from adherent cells, or cytokines produced by a T-cell hybrid EL4, resulted in an increased precursor frequency when LPS/DXS was added to the culture medium. Three macrophage cell lines, P388-D1, J774, and PU-5-IR, when added to purified B cells. augmented the autoantibody precursor frequency detected in vitro. This is strong evidence that potentially autoreactive B cells require one or more types of accessory cells in order to differentiate into autoantibody secretors during culture in vitro. Further, the results provide indirect evidence that interleukin 1 may be a crucial molecule in the differentiation of B cells.

Animals↗
PubMed1986

Limiting dilution analysis of age- and gender-related differences in autoantibody production against bromelain-modified RBC.

Cultured peritoneal cells from untreated mice, after 3 days of in vitro culture, produce autoantibodies against bromelain-treated isologous erythrocytes. The autoantibody response varies with both age and gender. The effects of age and gender were demonstrated by culturing peritoneal cells using limiting dilution techniques. In neonatal mice there were no precursor cells that differentiated into autoantibody secretors. Cells from female mice gave higher responses than cells from males, and the effect was more pronounced in cells from older mice and cultures to which lipopolysaccharide/dextran sulfate (LPS/DS) had been added. Various cell separation and cell mixing experiments indicated that a non-B-cell, nonadherent cell was involved in the higher autoimmunity detected in the presence of LPS/DS in female and older mice. It is thus possible that low autoimmune responses are due to the absence or unresponsiveness of accessory cells rather than potentially autoimmune B-cells.

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PubMed1985

Autoantibodies against mouse bromelain-modified RBC are specifically inhibited by a common membrane phospholipid, phosphatidylcholine.

Sera from mice injected 4 days earlier with lipopolysaccharide lysed mouse RBC treated with bromelain (brom). This lytic activity was totally inhibited by including phosphatidylcholine at final concentrations of about 2 micrograms/ml, or more, in the lytic mixtures. In contrast, the lytic activity of antibodies against rat RBC was not inhibited, even at concentrations of phosphatidylcholine up to 2.5 mg/ml. Various components of the phosphatidylcholine molecule, and other lipids including the closely-related molecule dipalmitoyl phosphatidyl-dimethyl-ethanolamine which is identical to dipalmitoyl phosphatidylcholine, except for the absence of a CH2 group on the polar head group, did not inhibit lysis by the autoantibodies. Autoantibodies against mouse brom RBC, but not antibodies against rat RBC, bound to, and could be eluted from, phosphatidylcholine molecules attached to an insoluble matrix. Liposomes of phosphatidylcholine prepared in the presence of phosphatidic acid or phosphatidylinositol did not inhibit the lysis of mouse brom RBC by autoantibodies to the same extent as liposomes of only phosphatidylcholine. This suggests that phosphatidylcholine is recognized by the autoantibodies only if presented in a certain configuration. We suggest that the function of these autoantibodies may be to facilitate the removal of membrane-damaged cells from the body. Such cells may arise by the process of ageing, or because of the effects of infectious agents such as viruses.

Animals↗
PubMed1984

Autoantibody-specific suppressor cells are elicited by membrane components of rat RBC independently of circulating autoantibodies but TNP-specific suppressors are not elicited by TNP-rat RBC.

Erythrocyte autoantibodies and autoantibody-specific suppressor cells are elicited in mice injected with rat erythrocytes. Here it is shown, first, that modified rat erythrocytes elicit suppressor cells independently of autoantibody production. Haemoglobin-free membranes of rat erythrocytes, but not soluble lysates of rat erythrocytes, elicited autoantibodies and suppressor cells. Solubilization of these rat erythrocyte membranes with solutions of Triton-X-114 produced membrane fractions which induced autoantibody-specific suppression in the absence of detectable autoantibody production. These results are compatible with the view that the activation of suppressor cells in this model involves recognition of structures on the rat erythrocytes. Secondly, trinitrophenyl (TNP) was attached to rat erythrocytes in various doses in attempts to make the immunogenicity of TNP similar to that of the cross-reactive determinants on rat and mouse erythrocytes, to determine whether TNP-specific suppressor cells were produced. The results show that at various doses of TNP, including those at which antibody production to TNP could not be detected, rat erythrocytes coated with TNP induced autoantibody-specific suppressor cells but not suppressor cells with specificity for antibodies to TNP.

Animals↗
PubMed1984

Peritoneal B cells differentiate without proliferation into autoantibody secretors under the influence of factors released by other cells.

After 3-5 days of in vitro culture, peritoneal cells from untreated C3H mice produce autoantibodies specific for autoantigens in the membranes of mouse erythrocytes. The autoantigens are exposed in vitro by treating mouse erythrocytes with the proteolytic enzyme bromelain. Limiting-dilution cell culture techniques were used to determine the frequency of the autoreactive B cells. Cells were cultured in Terasaki trays at 10-200 cells/well. Autoantibody production was assayed with an in situ plaque-forming cell (PFC) assay. On average, one autoantibody precursor cell was detected for every 150-200 peritoneal cells cultured. This precursor frequency was increased to 1 in 50 by the addition of lipopolysaccharide (LPS) and dextran sulphate (DS) to the culture medium. The addition of a culture supernatant from an EL4 lymphoma subline also induced a high proportion of the peritoneal cells to secrete autoantibodies. However, it was not possible to determine the frequency of PFC accurately because at limiting numbers of peritoneal cells the effects of EL4 affected more than one limiting variable. Significant cell division was observed in cultures to which LPS/DS had been added, in contrast to untreated cultures to which EL4 supernatant was added. The results show that high numbers of autoreactive B cells committed to self antigens are present in the peritoneal cavity and that these cells under the influence of appropriate cytokines can differentiate in vitro, even without proliferation, into autoantibody secretors. The cell type or types releasing the cytokines remain to be identified.

Animals↗
PubMed1984

Experimental erythrocyte autoimmunity. IV. Differential susceptibility to irradiation of suppressor cells for anti-rat RBC antibodies and suppressor cells for mouse RBC autoantibodies.

Mice injected with rat erythrocytes (RBC) produce RBC autoantibodies and antibodies against rat RBC. Transfer of spleen cells from autoantibody-producing mice to syngeneic recipients before the series of rat RBC injections causes a significant delay in autoantibody production although the response against rat RBC is elevated. Here it is shown that antibodies against rat RBC are markedly increased in recipients exposed to 350 rad of whole-body irradiation before transfer of spleen cells and the injections of rat RBC. In contrast, autoantibody production remained significantly suppressed. This shows that the anti-rat RBC response is regulated, at least in part, by cells in normal mice that are abrogated by 350 rad of whole-body irradiation.

Animals↗
PubMed1983

Experimental erythrocyte autoantibodies. V. Induction and suppression of red blood cell autoantibodies in mice injected with rat bromelain-treated red blood cells.

Mice injected with rat red blood cells (RBC), or rat bromelain-treated (brom) RBC, produce RBC autoantibodies and suppressor cells that specifically inhibit the autoimmune response without inhibiting the net production of antibodies against rat RBC. It has been investigated whether suppressor cells induced by injections of rat RBC are effective in preventing autoantibody production induced by rat brom RBC and vice versa. Autoantibodies were induced in C3H mice by weekly ip injections, each 0.2 ml, of a 6% suspension of rat RBC or rat brom RBC. Autoantibody production was assayed using Coombs' test. Suppressor cells were present in the spleens of mice positive in Coombs' tests and were shown by intravenous injections of 40 X 10(6) viable cells per mouse into untreated syngeneic mice 18 hr before the first injection of rat RBC or rat brom RBC. Autoantibodies eluted from mice positive in Coombs' tests after injections of rat RBC or brom RBC were absorbed by either type of rat RBC but not by RBC from sheep. This suggests that rat RBC and rat brom RBC display antigens that are similar, if not identical, to autoantigens on the mouse RBC. Spleen cells from mice injected with rat RBC suppressed autoantibodies induced by both rat RBC and rat brom RBC. In contrast, spleen cells from mice injected with rat brom RBC suppressed autoantibodies induced by rat brom RBC but not those induced by unmodified rat RBC. This differential suppression may be due to the removal from rat RBC, by bromelain, of a suppressor site and/or autoantigens of some specificities. Thus rat brom RBC may not induce the total range of specificities of autoantibodies, and of suppressor cells, induced by rat RBC.

Animals↗
PubMed1983

Non-H-2 genes involved in suppressing autoimmunity.

Peritoneal cells from C3H mice, after 4 days in culture, comprised a significantly higher number of cells secreting autoantibodies against mouse bromelain-treated red blood cells compared to cells from AKR mice. Females gave higher responses than males. The F1 hybrid of the two strains gave low-level responses similar to AKR mice. Mixtures of cells from males and females and from C3H and AKR mice did not give responses below those expected from the mixed population, which shows that cells from males and from AKR mice did not suppress the high-responder responder populations. However, cells from F1 mice significantly suppressed the response by C3H mice. The suppressor population was adherent to plastic and diminished by anti-Thy 1.2 treatment and complement. These results show that, within genetic constraints that include non-H-2 genes, suppressor cells likely to be T cells from a low-responder strain inhibit the development in vitro of an autoimmune response of a high-responder strain.

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