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Biomedical subjects

J D Mountz

Publications and source records attributed to J D Mountz.

At least 109 records · Page 6Linked to original sources

Production of transgenic mice and application to immunology and autoimmunity.

During the last decade, transgenic animal technology has assumed an increasingly important role as a critical tool in animal biology, biomedical research, and pharmaceutical development. This technology allows virtually any fragment of DNA large enough to contain an entire gene to become integrated into the germline of the recipient animal. The newly inserted DNA will be inherited like endogenous genes, and will be expressed as RNA and protein at tissue locations and abundance depending on regulatory elements attached to the coding DNA. It is possible to clone a particular gene, change a regulatory coding sequence, and reinsert the gene to determine the effect of the change on expression and function of the gene.

Allergy and Immunology↗

Studies of c-myb gene regulation in MRL-lpr/lpr mice. Identification of a 5' c-myb nuclear protein binding site and high levels of binding factors in nuclear extracts of lpr/lpr lymph node cells.

Lymph node T cells of MRL-lpr/lpr mice are characterized by the production of very large amounts of c-myb mRNA. To study the control of c-myb expression, a search was made for sites on the 5' c-myb gene which could bind regulatory proteins. DNase I digestion of nuclear chromatin uncovered four DNase I hypersensitive sites in the first intron of the c-myb gene, and a single site approximately 300 bp 5' to the initiation codon. Lambda exonuclease digestion of a 5'-myb fragment in the presence of nuclear extracts from either MRL-lpr/lpr PLN or EL-4 thymoma revealed stop sites approximately 300 bp 5' (-271 to -322) to the ATG initiation codon. DNase I footprint analysis demonstrated a guanine-cytosine enriched region of potential binding sites (-274 to -319) in the region of the stop sites and a fifth potential binding site closer to the initiation codon (-163 to -168). Specific gel shift bands were detected by a 5'-myb fragment (-346 to -155) with extracts from a number of different lymphoid cell lines and the appropriate specific and non-specific competitor DNA. The DNA giving rise to these gel shift bands encompassed the region defined by the stop site and footprinting studies. To determine whether or not the protein binding to the 5' c-myb gene at -274 to -319 was associated with increased c-myb mRNA, we studied nuclear extracts of several cell lines and compared the amount of binding to the amount of c-myb mRNA found on Northern analyses. Among the cell lines, there was a correlation between c-myb expression and the amount of the 5'-myb DNA binding protein. In addition, MRL-lpr/lpr lymph node cells had high c-myb expression and large amounts of the 5'-myb binding protein. This result suggests that the binding may play some role in the c-myb expression. Moreover, the most immature cell lines had the greatest amount of the binding factor, suggesting that its regulatory effect on c-myb expression might be important in early differentiation events.

Animals↗

Activation of CD 4-, CD 8- thymocytes with IL 4 vs IL 1 + IL 2.

Thymocytes from C57BL/6 mice were highly purified to obtain the CD 4-, CD 8- subpopulation which constitutes only 5% of all thymocytes. Substantial proliferation was induced in vitro with either IL-1 + IL-2 or with IL-4 in the presence of PMA. IL-1 and IL-2 synergized in inducing proliferation of these purified CD 4-, CD 8- thymocytes whereas neither synergized with IL-4. In order to determine whether stimulation with IL-1 + IL-2 acted via IL-4 or vice versa, cultures were treated reciprocally with affinity-purified anti-IL-2 or anti-IL-4 antibodies. Cultures with IL-4 were inhibited by anti-IL-4 but were unaffected by anti-IL-2. The CD 4-, CD 8- thymocytes cultured with IL-1 + IL-2 + anti-IL-2 were inhibited to baseline IL-1 stimulation. At low concentrations of IL-1 (1 U/ml) and IL-2 (100 U/ml), anti-IL-4 had no effect, whereas at higher levels of IL-1 (2 U/ml IL-1), and 100 or 200 U/ml IL-2, anti-IL-4 significantly reduced DNA synthesis. This result suggests that at higher concentrations the combination of IL-1 + IL-2 can induce cells to produce IL-4 which then contributes to overall proliferation. When CD 4-, CD 8- thymocytes were cultured with the low doses of IL-1 + IL-2 for 72 h, 62% expressed cell surface T3 complex (vs 11% at initiation) and 27% were F23.1+ (vs 5% at initiation). In contrast, culture with IL-4 led to no increase in numbers of T3+ cells and none were F23.1+; however, there was coexpression of Thy1 and 6B2 on 20% of cells at the end of culture (vs 4% at initiation). Thus, IL-1 + IL-2 causes expansion of a CD 4-, CD 8- thymocyte population expressing the alpha, beta-T cell receptor, whereas IL-4 induces cells to express a phenotype present in small numbers in the periphery of normal mice and in larger numbers in mice bearing the lpr gene.

Animals↗

Prevention of lymphadenopathy in MRL-lpr/lpr mice by blocking peripheral lymph node homing with Mel-14 in vivo.

MRL-lpr/lpr mice develop massive lymphadenopathy and autoimmunity. There is evidence that both migration and local proliferation contribute to the accumulation of Ly-2-, L3T4-, 6B2+ T cells in the peripheral lymph node (PLN). Mel-14 is an antibody which binds to the lymphocyte lymph node homing receptor (gp90Mel-14) and can block migration of lymphocytes to the PLN. Treatment of mice from birth to 11 wk of age with Mel-14 and another rat IgG2a mAb, 6B2, resulted in reduction (10- to 20-fold) in lymphadenopathy. Mel-14, but not 6B2, preferentially reduced the percentages of Thy-1+, 6B2+ lymphocytes in the lymph node. Treatment with a third antibody, anti-Ly-1, had no effect on lymphadenopathy. Mel-14 treatment resulted in diversion of the Ly-2-, L3T4-, 6B2+, gp90Mel-14 cells to the spleen and consequently induced marked splenomegaly. Thymocytes from MRL-lpr/lpr and MRL-+/+ mice were analyzed by two-color flow cytometry analysis after depletion of Ly-2+ and L3T4+ T cells. There was no difference in the percent of Ly-2-, L3T4-, 6B2+, gp90Mel-14 positive thymocytes comparing these two strains. Mel-14 treatment did not alter Ig levels or autoantibody production. These studies suggest Mel-14 reduced lymphadenopathy by interfering with homing to PLN, whereas 6B2 may have interfered with marrow production of precursor cells or killed 6B2+ cells after they exited the marrow. The data are consistent with the idea that lymphadenopathy occurs in MRL-lpr/lpr mice due to increased homing gp90-Mel-14 T cells to the PLN and that gp90Mel-14 is a necessary receptor for the abnormal 6B2+ T cells.

Animals↗

The expression and modulation of IL-1 alpha in murine keratinocytes.

Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low [Ca2+] tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high [Ca2+] media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low [Ca2+] conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes. Thus LPS, UV, and cell differentiation state have a significant effect on expression of IL-1 alpha in murine keratinocytes.

Animals↗

Characterization and differentiation of CD4-, CD8- thymocytes sorted with the Ly-24 marker.

Heterogeneity within the CD4-, CD8- thymocyte population was explored by flow microfluorometry on thymocytes from 6-wk-old female C57BL/6 mice. Double negative cells were obtained by twice killing thymocytes with anti-CD4 and anti-CD8 antibodies. The resultant population lacked CD4, CD8, and Ig on cell surfaces; it contained cells bearing Ly-24 (27%), Ly-6C (6%), and Ly-5 (B220) detected by 6B2 (6%). These markers are the same as those characteristic of lpr/lpr T cells; they also are found on bone marrow cells. In order to investigate the maturational pathway of CD4-, CD8- thymocytes, such cells were cultured in vitro for 6 days with phorbol myristate acetate + calcium ionophore. There was a marked increase in cells bearing Ly-24 with time; by 6 days essentially all bore Ly-24. A lesser increase (to 15%) in 6B2 + Thy-1 positive cells was observed. Small numbers of cells bearing CD4 and/or CD8 also were found after 6 days in vitro. In additional studies, CD4-, CD8- cells were first sorted with respect to Ly-24 and then cultured with phorbol myristate acetate + calcium ionophore. Ly-24+ cells proliferated vigorously and formed clusters whereas Ly-24- cells did not. The former gave rise to large numbers of CD4+, CD8+ cells; the latter exhibited little differentiation. These studies demonstrate substantial heterogeneity within the CD4-, CD8- thymocyte population. Use of the markers Ly-24, Ly6C, and 6B2 allows a subdivision of such progenitor thymocytes. Different stages of maturation as well as possible lineages of cells may be investigated by combining such hemopoietic cell surface markers with in vitro culture.

Animals↗

Polyclonal activation of the murine immune system by an antibody to IgD. VIII. Stimulation of IgD secretion.

The low levels of serum IgD found in mice and the lack of a typical DNA switch sequence between C delta and C mu raise the possibility that the generation of murine IgD-secreting cells results from a chance "mistake" rather than a controlled process. The recent observation that injection of mice with purified IgD upregulates IgD receptor expression on helper T cells and enhances the ability of these T cells to induce B cells to differentiate into antibody secreting cells led us to look for evidence of controlled differentiation of B cells into IgD-secreting cells. To do this, we injected mice with a goat antibody to IgD (GaM delta), because this antibody stimulates large increases in IgM, IgG1, IgG2a, and IgE secretion. Mice injected with GaM delta demonstrated a large increase in splenic content of mRNA specific for the secreted form of delta-chain, as well as a greater than 100-fold increase in the percentage of splenic IgD-containing plasmablasts. The secretory IgD response was totally T-dependent. Production of the secretory form of IgD was not seen until 7 days after GaM delta injection, and peaked sharply on day 8, whereas by day 6 IgM secretion had already peaked and IgG1 and IgG2 secretion had attained substantial levels. This observation suggests that: 1) either cells that synthesize large quantities of the secretory form of delta-chain, unlike cells that synthesize large quantities of the secretory forms of gamma-, epsilon-, or alpha-chains, do this without deleting C mu, or, despite the absence of a typical DNA switch sequence between C mu and C delta, controls must exist to effect the C mu deletion and VDJ-C delta joining; and 2) if secreted IgD has a role in the regulation of a humoral immune response it most likely is involved in later processes, such as memory cell generation or response termination, rather than in relatively early processes, such as helper T cell activation.

Animals↗

Polyclonal activation of the murine immune system by a goat antibody to mouse IgD. IX. Induction of a polyclonal IgE response.

The possibility that injection of mice with an affinity-purified goat antibody to mouse IgD (GaM delta) that stimulates polyclonal IgG1 secretion might also stimulate differentiation of B cells into IgE-secreting cells was suggested by the observation that such treatment induces T cells from those mice to secrete a lymphokine, B cell stimulatory factor 1 (BSF-1), that can stimulate both IgG1 and IgE secretion in vitro. Studies described in this paper show that injection of BALB/c mice with 200 to 3200 micrograms of GaM delta greatly increased the quantity of splenic epsilon chain-encoding mRNA, the number of spleen cells with cytoplasmic IgE, and the concentration of serum IgE 7 days after injection. Serum IgE levels obtained in these mice were approximately 100 times baseline levels and were comparable with those found in mice infected with the nematode parasite Nippostrongylus brasiliensis, but were approximately 2000-fold less than the peak serum IgG1 levels induced by GaM delta injection. Both IgE and IgG1 secretion in GaM delta injected mice were T dependent (blocked by anti-L3T4 antibody). These observations are consistent with the hypothesis that BSF-1 may play a role in the in vivo stimulation of IgE secretion and provide an easy to apply model for the investigation of in vivo regulation of IgE responses.

Animals↗

CS-A therapy in MRL-lpr/lpr mice: amelioration of immunopathology despite autoantibody production.

MRL-lpr/lpr mice spontaneously develop massive T cell lymphadenopathy, autoantibodies, and immune-mediated pathology. These mice are thought to be models of various human autoimmune diseases, including systemic lupus, Sjogren's syndrome, and rheumatoid arthritis. We have used cyclosporin A (CS-A) treatment as a tool by which the mechanisms of immune-mediated pathology might be dissected. CS-A was used because of its known preferential inhibition of T cell function and the marked expansion in MRL-lpr/lpr mice of an unusual L3T4-, Lyt-2-, 6B2+ T cell population. CS-A prevented lymphadenopathy and expansion of L3T4-, Lyt-2-, 6B2+ T cells in the peripheral lymph nodes, and also in the thymus. The increased expression of the c-myb and T cell receptor beta-chain genes associated with these unusual cells was also corrected. The finding of increased numbers of L3T4-, Lyt-2-, 6B2+ thymocytes in untreated mice suggests abnormal intrathymic differentiation in lpr/lpr mice, a defect that was corrected by CS-A. Treated mice had a marked decrease in arthritis and glomerulonephritis and significantly prolonged survival. These beneficial effects of CS-A occurred despite a lack of reduction in antibodies reactive with DNA, circulating immune complexes, rheumatoid factor titers, or immunoglobulin concentrations. These results demonstrate that the B cell hyperactivity of MRL-lpr/lpr mice can proceed without the T cell proliferative disease.

Animals↗

Subpopulations of CD4-, CD8- thymocytes.

A population of highly purified CD4-,CD8- thymocytes was analyzed by both flow microfluorometry and in situ hybridization in an attempt to further elucidate thymocyte subpopulations. The double-negative cells had a marked increase in expression of the c-myb and T cell gamma genes relative to unseparated thymocytes. Approximately 27% were Ly-24+, 8% Ly-6C+ and 6% 6B2+. All of the Ly-6C+ cells were also Ly-24+. A small population (6%) of the CD4-,CD8- thymocytes had surface expression of the T cell receptor for antigen (F23.1); all were bright Ly-1+ and half were Ly-24+. These studies demonstrate that there are further subdivisions of the CD4-, CD8- thymocytes based upon cell surface expression of markers previously found on bone marrow cells and their non-T cell progeny. Studies are in progress to determine whether these represent different stages of activation-maturation or different lineages of cells.

Animals↗

IL-2 modulation of murine T-cell oncogene expression.

C-myb, a cellular oncogene associated with normal thymic development, was found to be highly expressed in four interleukin 2 (IL-2)-independent T-cell lines, but not in two of three IL-2-dependent T-cell lines. The IL-2-dependent lines, HT2 and CTLL-2, were found to have low levels of c-myb mRNA in the presence of IL-2. However, short-term IL-2 depletion resulted in at least fivefold increases in c-myb message. Add-back of IL-2 after 30 hr IL-2 depletion of CTLL-2 cells resulted in return to baseline low-level c-myb mRNA. Expression of the oncogenes myc, bas, raf, and abl as well as the T-cell genes Thy-1 and CT beta did not parallel that of c-myb. These studies indicate that removal of a growth factor can result in increased levels of a specific cellular oncogene and that two nuclear protooncogenes (c-myb and c-myc) are expressed differentially during cell growth. These results may help to explain aspects of intrathymic T-cell differentiation where there is very high c-myb expression in the face of limiting amounts of growth factors such as IL-2.

Animals↗

Genetic and molecular genetic studies of murine and human lupus.

Mice and humans with systemic lupus erythematosus (SLE) have been studied with regard to cellular, genetic and molecular genetic abnormalities. B cell hyperactivity and autoantibody production are the hallmarks of this illness. In humans with SLE, there is increased stem cell, B cell precursor and B cell proliferation. The same is true of NZB mice. In lpr/lpr and gld/gld mice, marked expansion of a subpopulation of T cells allows extrathymic terminal T cell maturation and secondary B cell hyperactivity. Androgens suppress these processes and polyclonal immune activators accelerate them. Three types of genes are identified: inducing genes, accelerating genes and background genes. These give rise to abnormal expression of various cellular oncogenes, T cell receptor genes and immunoglobulin genes. The data suggest that abnormal immune regulation plays a critical role in the development of SLE, with polyclonal B cell activation being common to both mice and humans with SLE. Different genetic and cellular abnormalities underlie the ultimate syndrome, the common denominator, generalized autoimmunity, that we call SLE.

Animals↗

T cell receptor gene expression in autoimmune mice.

Autoimmunity in mice with the lpr/lpr and gld/gld genotypes is accompanied by profound lymphadenopathy characterized by the presence of a massive expansion of an unusual T cell subset. The abnormal lymph node T cells were found to express TcR beta and TcR alpha transcripts of expected sizes. There was a 10-fold increase in the 1.3-kb TcR beta transcript and a twofold increase in TcR alpha gene expression, even though Thy-1 expression was in general similar to controls. A study of T cell receptor expression during ontogeny failed to reveal any striking differences between lpr/lpr and congenic mice. There was a strong correlation between TcR beta expression and c-myb expression; however, there was no necessary association of TcR beta and c-myb expression when various T cell lines were examined. Background genes were found to influence the expression of T cell receptor genes in lpr/lpr mice. AKR-lpr/lpr lymph node cells, but not cells from other lpr/lpr mice or AKR +/+ mice, had the predominance of the 1.0-kb TcR beta transcript, which represents the nonfunctional D-J TcR beta rearrangements. Lymph nodes from MRL-lpr/lpr mice, but not C57BL/6-lpr/lpr or AKR-lpr/lpr mice, were found to express small amounts of the TcR gamma transcript. In addition, MRL-lpr/lpr but not C57BL/6-lpr/lpr mice had an age-related decrease in thymic TcR beta expression along with a decrease in thymic c-myb expression. The current study extends the characterization of T cell gene expression abnormalities in peripheral T cells of gld/gld and lpr/lpr and describes certain similarities of these cells to immature thymocytes at a molecular level. Furthermore, it illustrates the complex interactions between "background genes" and genes responsible for lymphoproliferation, which in concert lead to specific molecular and cellular abnormalities.

Animals↗

Oncogene expression in autoimmune and normal peripheral blood mononuclear cells.

PBMC from patients with autoimmune diseases and from normal controls were studied for the expression of several cellular oncogenes. Gene expression was assessed by Northern blot analysis of poly(A)+ RNA obtained from leukapheresis samples. Patients with SLE expressed significantly more c-myc protooncogene RNA than did normal controls. Increased expression of the N-ras protooncogene was found in that subset of patients whose autoimmune disease was very active. Cells from individuals with SLE, but not from those with other autoimmune illnesses, showed significantly decreased levels of the c-myb and c-fos protooncogenes. To examine the implications of these findings, B and T cells were purified from apheresis samples donated by normal volunteers. When mitogen was used to activate the B cells in vitro, their pattern of protooncogene expression changed to resemble that found in freshly isolated cells from lupus patients. These results suggest that the differences detected in the expression of protooncogenes by patients with SLE may be due to the abnormal activation of their B cells in vivo. The pattern of protooncogene expression found in patients with other autoimmune illnesses is consistent with the activation of additional cell types in those diseases.

Autoimmune Diseases↗

Pks, a raf-related sequence in humans.

A human fetal liver cDNA library was screened at reduced hybridization stringency for v-raf-related sequences. In addition to the expected c-raf-1 cDNA, a second sequence was isolated. Comparison of the second gene (pks) to the other raf-related sequences revealed nucleotide homologies of 71%. The predicted amino acid sequence of the kinase domain is sufficiently similar to that of v-raf to suggest that pks may encode a polypeptide that exhibits serine/threonine kinase activity. The expression of pks mRNA (2.7 kilobases long) is elevated in peripheral blood mononuclear cells isolated from two patients with angioimmunoblastic lymphadenopathy with dysproteinemia, a disease in which autoantibodies are produced following the lymphoproliferative activation of B cells. Analysis of somatic cell hybrids for segregation of the pks locus revealed the presence of an additional locus closely related to the pks sequence.

Amino Acid Sequence↗