Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture.

Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.

Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases.

When cells are stimulated by mitogens, extracellular signal-regulated kinase (ERK) is activated by phosphorylation of its regulatory threonine (Thr) and tyrosine (Tyr) residues. The inactivation of ERK may occur by phosphatase-mediated removal of the phosphates from these Tyr, Thr or both residues together. In this study, antibodies that selectively recognize all combinations of phosphorylation of the regulatory Thr and Tyr residues of ERK were developed, and used to study the inactivation of ERK upon mitogenic stimulation. We found that inactivation of ERK in the early stages of mitogenic stimulation involves separate Thr and Tyr phosphatases which operate differently in the nucleus and in the cytoplasm. Thus, ERK is differentially regulated in various subcellular compartments to secure proper length and strength of activation, which eventually determine the physiological outcome of many external signals.

Detection of ERK activation by a novel monoclonal antibody.

The mitogen-activated protein kinase, ERK is activated by a dual phosphorylation on threonine and tyrosine residues. Using a synthetic diphospho peptide, we have generated a monoclonal antibody directed to the active ERK. The antibody specifically identified the active doubly phosphorylated, but not the inactive mono- or non- phosphorylated forms of ERKs. A direct correlation was observed between ERK activity and the intensity in Western blot of mitogen-activated protein kinases from several species. The antibody was proven suitable for immunofluorescence staining, revealing a transient reactivity with ERKs that were translocated to the nucleus upon stimulation. In conclusion, the antibody can serve as a useful tool in the study of ERK signaling in a wide variety of organisms.

Natural autoantibodies in sera of patients with Gaucher's disease.

Gaucher's disease (GD) is associated with hyperactivity of the immune system, which manifests by polyclonal hypergamma-globulinemia and an increased incidence of monoclonal gammopathies in GD patients. We analyzed sera of 43 patients with GD for the presence of autoantibodies against 14 autoantigens. The results demonstrated a significant increase in the incidence of all autoantibodies tested, ranging from 11% for anti-RNP, pyruvate dehydrogenase (PDH), and DNA antibodies to 57% for rheumatoid factor. The autoantibodies were of all three isotypes, namely, IgG, IgM, and IgA. There was no correlation between the levels of immunoglobulins in the serum and the titer of autoantibodies found. Immunization of naive mice with a pool of purified anti-DNA antibodies form GD patients did not result in induction of experimental systemic lupus erythematosus (SLE), suggesting that they may represent natural autoantibodies that are not pathogenic. In conclusion, we found high titers of natural, polyspecific, nonpathogenic autoantibodies in the sera of GD patients.

Antimitochondrial (pyruvate dehydrogenase) antibodies in leprosy.

Sera from 69 patients with leprosy but without liver involvement were assayed for the presence of mitochondrial pyruvate dehydrogenase (PDH)-specific autoantibodies by enzyme-linked immunoabsorbent assay (ELISA), immunoblotting using PDH as an antigen and by enzymatic inhibition test. Twenty-seven of the leprosy serum samples (39.1%) were found to react with PDH by ELISA. However, unlike sera from primary biliary cirrhosis (PBC) patients, none of these were able to inhibit the PDH enzymatic activity. By immunoblotting, it was found that only 2 of the 27 positive sera recognized the 74-kD protein of the PDH complex, which is recognized by sera of most PBC patients. The antimitochondrial antibodies in lepra most probably recognize different epitopes than those in PBC. These findings may indicate that anti-PDH autoantibodies in patients with leprosy may arise by polyclonal B cell stimulation and may represent natural anti-PDH autoantibodies.

A rheumatoid factor transgenic mouse model of autoantibody regulation.

To address whether B cells expressing a disease-associated autospecificity are regulated in normal mice, we have established a rheumatoid factor (RF) transgenic model of autoimmunity, using V genes derived from an IgA anti-IgG2a RF isolated from an autoimmune MRL/lpr mouse. As we wished to study induction of tolerance during B cell development, we cloned the VH gene into an IgM expression vector. The RF we chose binds only IgG2a of the 'a' allotype (IgG2a) but not IgG2ab allowing us to produce transgenic animals on IgHa and IgHb backgrounds, which either express or lack the self-antigen. Two transgenic lines were studied. Using mice which lack the self-antigen, we show by fluorescence activated cell sorting and hybridoma analysis that the H and L transgenes are expressed to the exclusion of endogenous genes in most splenic B cells. In spite of good allelic exclusion, transgenic mice which are genetically capable of expressing IgG2aa have reduced but significant (approximately 50 micrograms/ml) serum levels. Nonetheless, the frequency and numbers of transgene-expressing B cells in peripheral lymphoid organs of such mice which have the self-antigen are similar to those which lack it (IgHb mice). Thus, B cells expressing an anti-self IgG2a surface receptor can develop in this system. Whether such B cells are anergic or otherwise regulated in autoantigen-expressing mice is discussed.

Quantitative analysis of bone marrow thymic progenitors in young and aged mice.

Our studies on the capacity of bone marrow (BM) to generate T lymphocytes in aging have revealed that under the competitive conditions of thymic reconstitution, cells of aged mice are significantly inferior to those of the young. The present study was designed to further investigate the basis of this age-related change. Two mechanisms were considered: (a) The potential of BM-derived T cell precursors from aged mice to proliferate and differentiate in the thymic microenvironment is impaired. (b) The frequency of T cell precursors is reduced in BM of aged mice, thus affecting their ability to compete efficiently in reconstituting the thymus. These possibilities were studied in vitro by colonizing thymocyte-depleted fetal thymic lobes with BM cells from aged (24-month) and young (3-month) C57BL/6 mice. By determining the cell cycle duration of BM-derived cells which have seeded the thymic lobes, we found that cells originating from aged mice proliferate in the thymus at the same rate as those from young mice. Reconstitution with limiting numbers of BM cells indicated that the frequency of thymic progenitors in the BM is significantly reduced in aged as compared to young mice. We thus conclude that aging is associated with a quantitative reduction in the frequency of thymic progenitors in the BM.

Altered VH gene segment utilization in the response to phosphorylcholine by aged mice.

Newly generated bone marrow B cell precursors of aged BALB/c mice, stimulated in splenic fragment cultures, display a markedly increased frequency of phosphorylcholine (PC)-responsive cells. This increased frequency is found for both precursors that utilize VHS107, a phenotype common to essentially all PC-specific B cells of young mice, and, surprisingly, for precursors that utilize VH genes other than VHS107. PC-specific hybridomas derived from bone marrow cells of aged mice utilize members of at least three VH gene segment families that have never been observed in PC responses of young mice. The ability of aged but not young mice to generate these unique PC-specific clonotypes may be evidence for constraints on V region utilization during repertoire development in young adults and has important implications for aging-associated changes in immune responsiveness.

The role of IL-4 in the generation of B lymphocytes in the bone marrow.

It has been indicated that IL-4 supports the maturation of pre-B cells to B lymphocytes. The present study was designed to investigate the mechanism by which IL-4 influences this maturational process. In order to determine whether IL-4 acts directly on pre-B cells, we sorted out B220+, sIg- cells from bone marrow of young adult (C3H X C57BL/6)F1 mice. These purified populations of pre-B cells (greater than 95%) were incubated with and without 4 to 200 U/ml of rIL-4, and the generation of new B lymphocytes in these cultures was followed for several days. We found that the frequency of newly generated B lymphocytes (%sIg+) was similar in control and in IL-4-containing cultures. However, the total number of B lymphocytes was significantly higher in IL-4-containing cultures. This high number of B lymphocytes was a result of an increased survival of cells in IL-4-containing cultures. The effect was IL-4 specific because anti-IL-4 antibodies completely prevented this phenomenon. We thus conclude that IL-4 does not induce the maturation of pre-B cells but may be important in the process of B lymphocytes generation by providing a signal for survival of these cells.

Age-related changes in the capability of the bone marrow to generate B cells.

The capability of the bone marrow (BM) to generate new B cells in aging was studied in vitro. BM cells from old (26 to 30 mo) or young (3 mo) BALB/c and (C3H/eB x C57BL/6)F1 mice were depleted of mature B cells and these surface Ig (sIg) BM cells were incubated in culture for 3 days. The frequency of newly generated B cells in these cultures was determined by assessing the frequency of slg+ cells and of B cells forming colonies in agar and by assaying the proliferative capacity of these newly generated B cells after stimulation by LPS. We found that BM cells from aged mice are significantly inferior to young ones in their capability to generate new B cells in culture. By mixing old and young slg- BM cells, we found that, in general, this reduction was not caused by a suppressive effect of T cells or of any other cells, but rather to lack of some sort of supportive cell or factor in the aged BM. In addition, we found that the frequency of cells expressing the B220 surface molecule, a B lineage-specific marker, is significantly reduced in aged BM. These results indicate that a quantitative decrease in B cell progenitors combined with changes in cell populations that are important in supporting B cell generation contribute to the age-related decrease in the capacity to generate B cells.

Age-related changes in the capacity of bone marrow cells to differentiate in thymic organ cultures.

The capacity of the bone marrow to give rise to T cells in advanced age was studied in vitro by reconstituting fetal thymic lobes from 14-day C57BL/Ka (Thy-1.1) mice with bone marrow cells from old (24-month) or young (3-month) C57BL/6 (Thy-1.2) mice. The use of these congenic strains enabled distinguishing between donor and host contribution to the developing T cells. We found that bone marrow cells from aged mice maintained their capacity to reconstitute fetal thymic explants and to differentiate into various T-cell subsets as assessed by distinct T-cell-specific surface markers (Thy-1, Lyt-1, Lyt-2, and L3T4) and functions (concanavalin A-induced proliferative and cytotoxic responses). However, when mixtures of old and young bone marrow cells reconstituted fetal thymic explants, the cells of old mice were less efficient than those of young in their capacity to give rise to T cells. These results indicate that bone marrow cells from aged mice can reconstitute the thymus and differentiate into T cells; however, their reconstituting capacity is inferior to that of bone marrow cells from young mice.

Ontogeny of T cells: development of pre-T cells from fetal liver and yolk sac in the thymus microenvironment.

The patterns of development of T cells from the very early stem cells that settle in the embryonic thymus have been studied. For this purpose, mouse embryonic thymuses (14 days) depleted of thymocytes were reconstituted with hemopoietic stem cells from fetal liver (FL) and yolk sac (YS) and T-cell development was followed in vitro in organ culture. It was found that cells derived from FL and YS of 10- to 14-day-old embryos were capable of reconstituting depleted thymic explants and exhibiting membrane markers in a pattern similar to that of thymocytes developing in intact thymic explants. Furthermore, these cells responded to concanavalin A in proliferative and cytotoxic assays as measured by limiting-dilution analysis. Thus, lymphohemopoietic stem cells emerging in the embryo prior to thymus lymphoid development are capable of differentiation in the thymus microenvironment into T cells, identified by phenotypic markers and functions that are characteristic of cells developing in the intact embryonic thymus.

T cell involvement in the decrease of antigen-responsive B cells in aged mice.

The role of T cells in the reduced frequency of splenic B cells specific for several antigens in aged mice was studied by assessing B cell responsiveness in (a) aged nude mice and (b) irradiated young mice repopulated with splenic B cells or with Ig- bone marrow cells from young mice and T cells from aged vs. young mice. Using the fragment culture technique to assess B cells specific for 2,4-dinitrophenyl (DNP) and for (4-hydroxy-3,5-dinitrophenyl) acetyl, we found that the frequency of responsive splenic B cells in aged BALB/c nude mice was very similar to that of young nude mice. In addition, we found that in chimeric mice constructed with either bone marrow or splenic B cells from young mice and T cells from aged mice the frequency of DNP-specific splenic B cells was significantly lower than that in control chimeras constructed with T cells from young mice. These results indicate that T cells from aged mice can down regulate B cell responsiveness and that a mature, naive B cell may be its possible target. The results of both experimental approaches are consistent with a role for T cells in the regulation of responsive B cells in aging.

The frequency and fine specificity of B cells responsive to (4-hydroxy-3-nitrophenyl)acetyl in aged mice.

The B-cell response to NP-Hy of two murine strains has been analyzed in order to evaluate the affects of aging on B-cell repertoire expression. The results indicate that for both BALB/c mice (Igha) and B10.D2 mice (Ighb) the frequency of (4-hydroxy-3-nitrophenyl)acetyl (NP)-responsive splenic B cells is approximately twofold lower, on a per B cell basis, in aged mice as compared to young adults. However, as with previous assessments of the response to DNP-Hy in aged mice, the frequency of newly generated surface immunoglobulin negative bone marrow precursor cells specific for NP in aged BALB/c mice is the same as in young mice. The decrease in frequency of responsive splenic B cells is not accompanied by a measurable decrease in repertoire diversity or changes in clonotype distribution as assessed by representation of kappa vs lambda light chain-bearing antibodies, binding of monoclonal antibodies to a panel of analogues of NP, and the proportionate representation of B10.D2 monoclonal antibodies that bear NPb idiotypic determinants. By these criteria it appears that down-regulation of B cells as they mature and emerge from the bone marrow of aged mice is pan-specific and does not disproportionately affect B cells of a predominant clonotype family. Consistent with other investigations which have demonstrated differences in secreted antibodies of immunized aged vs young mice, we have observed that 4 weeks after immunization of B10.D2 mice with NP-BSA, the frequency of newly generated secondary B cells is lower in aged than in young mice and the generation of lambda-bearing secondary precursor cells is particularly low. Thus, clonotype-specific down-regulation may play a role in shaping the B-cell repertoire subsequent to immunization of aged mice.

B cell repertoire diversity to PR8 influenza virus does not decrease with age.

The affect of aging on the B cell repertoire was investigated at the clonal level by studying the response of 24- to 26-mo-old BALB/c mice to the PR8 influenza virus (H1N1). By using the splenic fragment culture technique, it was found that the frequency of B cells specific for either the intact virion or the HA does not change with age. The anti-HA monoclonal antibodies obtained from culture were additionally analyzed for fine specificity by binding in RIA to a panel of six H1 variant viruses. This analysis showed a considerable similarity in the distribution and diversity of reactivity patterns between monoclonal antibodies derived from splenic B cells of young vs aged mice. These findings indicate that repertoire expression per se may not be truncated in aged mice, and imply that reductions in the diversity of serum antibodies in aged mice may be due to environmental regulatory mechanisms.

T-cell cytotoxicity and aging: differing causes of reduced response in individual mice.

Specific T-cell cytotoxic responses to allogeneic and hapten-modified syngeneic cells decrease with age. In order to determine the causes of these reduced T-cell cytotoxic responses, spleen cells from individual young and senescent C57BL/6J female mice were mixed in various proportions in culture with either X-irradiated BALB/c spleen cells or trinitrophenyl-modified syngeneic cells and the resultant cytotoxic responses determined in comparison to those of spleen cells from young and old mice stimulated alone. In both allogeneic and hapten-modified syngeneic cytotoxicity, it was found that a low percentage of the aged mice suffered from decreased helper-cell activity or from increase of suppressor activity, while the majority of mice showed no synergy, positive or negative, with the cells from the young donor. Studies of interleukin-2 (IL-2) activity were performed on conditioned medium from spleen cells from mice of various ages cultured for 24 h with concanavalin A. Those preparations from senescent mice that showed reduced IL-2 activity did not contain activity suppressive or competitive to IL-2 produced by spleen cells from young mice. Limiting dilution of spleen cells from mice of various ages in the presence of semi-allogeneic stimulatory cells and subsequent assay of the resultant allogeneic cytotoxicity provided a measure of the frequency of cytotoxic units. Parallel experiments in which crude IL-2 was added to the limit dilution cultures provided a measure of the frequency of cytotoxic cell precursors. Once again in these experiments, individual senescent mice demonstrated different defects. Three different types of age-related defects were observed. Certain aged mice were devoid of detectable cytotoxic units and cytotoxic T-lymphocyte precursor at the cell dilutions used. Other senescent mice demonstrated a very low frequency of cytotoxic units (approximately 1/40 000) as compared with young mice (approximately 1/5 000), and the addition of crude IL-2 to cultures from these mice did not improve reactivity. A third group of old mice, those with a moderate age-related decrease in the frequency of cytotoxic units (approximately 1/12 000), demonstrated a cytotoxic T-lymphocyte precursor frequency in the presence of crude IL-2 which was comparable to that of young mice (approximately 1/1000).

Antigen responsiveness of the mature and generative B cell populations of aged mice.

The deficit of humoral immune responsiveness associated with aging was investigated at the level of individual antigen-specific B cells. It was found that mature dinitrophenyl (DNP)-responsive B cells isolated from the spleen of aged mice gave rise to clones of antibody-forming cells that were normal with respect to both the amount and relative affinity of anti-DNP antibody produced. However, although the proportion of immunoglobulin-bearing cells in the spleen of aged mice was normal, the proportion of cells that responded to T cell dependent DNP-specific stimulation (1.1 per 10(6) injected cells) was significantly lower than the proportion that responded when cells were obtained from the spleen of young mice (2.3 per 10(6) injected cells). To examine the origin of this diminution in antigen-responsive B cells, the responsiveness of precursor cells from the B cell generative pool isolated as the surface immunoglobulin negative (sIg-) cells within the bone marrow was evaluated. The frequency of DNP-responsive cells in both intact bone marrow cell suspensions and the sIg- subpopulation was not significantly different when such cells were isolated from aged vs. young individuals. Thus, it would appear that among the immunologic deficits associated with aging is a decrease in the proportion of antigen-responsive B cells, which is associated with maturation of B cell clones in the aged environment and occurs during the migration of cells from the bone marrow to the spleen.