Pre-pre-B cells containing only cytoplasmic B220 can be induced by dendritic cells and T cells to differentiate in vitro.

We have derived from spleens of nude mice early B lineage cells that were phenotypically compatible with a pre-pre-B cell stage of differentiation. Although these cells containing large basophilic granules had the B lymphocyte antigen B220, in the cytoplasm, they had no surface B220, no cytoplasmic or surface immunoglobulin heavy or light chains, no surface Thy-1, and no surface Ia. In addition, they appeared to have little or no heavy chain gene rearrangements, including the D to J that occurs on both chromosomes prior to the VH rearrangement that forms the code for the C mu heavy chain polypeptide. Cells at even this early stage of differentiation could be induced by DC-T to express B220 on the surface and to synthesize and then to secrete immunoglobulins. These phenotypic changes were associated with a morphologic change in the cells to a lymphoblastoid appearance. Different patterns of immunoglobulin secretion resulted when pre-pre-B cells were cocultivated with DC-T from different tissues; SP DC-T induced the secretion of only IgM, PP DC-T induced the secretion of IgM as well as IgG and IgA. The early inductive event(s) appeared to occur during cell-cell contact in aggregates of the inducing DC-T and the pre-pre-B cells.

Exquisite sensitivity of dendritic cells to ultraviolet radiation and temperature changes.

Recent data suggest that dendritic cells (DC) are the critical passenger leukocytes in allograft rejection. Moreover, previous studies suggest that ultraviolet radiation (UVR) abrogates many in vivo and in vitro immune responses in which DC function as potent accessory cells (AC); however, the mechanism(s) underlying the suppressive effect of UVR on these responses is unclear. To address this mechanism, the hypothesis was tested that loss of DC viability (hence function) accounts for the suppressive effect of UVR on these responses. To this end, in vitro effects of UVR on murine splenic DC viability were addressed using two types of UVR (ultraviolet B [UVB] and ultraviolet C [UVC]) over a UVR dose range of 0-864 J/m2. DC viability was exquisitely sensitive to UVR when compared with other AC populations and UVC was 4-fold more effective in decreasing DC viability than UVB when doses of equal energy were compared. It was found that both UVR types induced marked decreases in DC viability beginning 4-6 hr post-UVR-treatment, that UVR- and non-UVR-induced death were temperature-dependent, and that decreases in DC viability induced by UVR were compatible with interphase death. Our findings indicate that DC are sensitive to temperature changes and exquisitely sensitive to UVR, and suggest that UVR-induced abrogation of murine immune responses is likely attributable to UVR-induced DC death.

Many different levels of maturation and commitment are present in nontransformed cells identified serologically as pre-B cells.

The pre-B cell stage of B lymphocyte development is characterized by the presence of immunoglobulin heavy chains of the IgM isotype in the cytoplasm and no other heavy or light chains in the cytoplasm or on the surface. We established several cell lines that were identical in their serologically defined pre-B cell phenotypes and in their dependence upon interleukin 3 for growth, but which differed in their levels of cytoplasmic RNA from immunoglobulin constant region genes, in their rates of differentiation in vitro, and in the isotype profile of the antibodies that they secreted upon differentiation. The two cell lines that we have analyzed in detail, PF1 and PF1C, both contained RNA from the C mu and C delta heavy chain genes and from both the C kappa and C lambda light chain genes, even though they were not producing detectable polypeptide products from the C delta, C kappa, or C lambda genes. However, PF1C had higher levels of C gamma and C alpha RNA transcripts and differentiated in vitro under the influence of dendritic cells and T lymphocytes (DC-T) more rapidly than did PF1. If the DC-T were derived from spleens, all cell lines secreted only IgM. However, under the influence of DC-T from Peyer's patches PF1C secreted predominantly IgM, PF1 secreted primarily IgA, and a third line, PF3, secreted primarily IgG. Therefore, within the population of cells described as pre-B cells on the basis of their immunoglobulin gene polypeptide products, there are subpopulations that probably represent different levels of maturation and different levels of commitment to particular pathways of B lymphocyte development.

Enhancement of IL-2-induced T cell proliferation by a novel factor(s) present in murine spleen dendritic cell-T cell culture supernatants.

The mechanism(s) underlying the potent accessory cell function of dendritic cells (DC) remains unclear. The possibility was considered that a soluble factor(s) released during the interaction of DC and T cells might contribute to the potent T cell activating function of DC. Culture supernatants were generated from mixtures of murine spleen DC and periodate-treated spleen T cells and were examined for the presence of known cytokine activities and factors capable of enhancing T cell responsiveness to IL-2. Serum-free supernatants from 24 h DC-T cell co-cultures exhibited high levels of IL-2, detectable levels of IL-3, and negligible levels of IL-1, -4, -5, -6, and TNF. A factor(s) was also identified with an apparent Mr of 12.5 to 17.0 kDa, henceforth designated IL-2 enhancing factor (IL-2EF), which enhanced the IL-2-induced proliferation of murine thymocytes, CTLL, and HT-2 cells by approximately three- to fourfold. This enhancement was also observed in the presence of neutralizing antibodies to murine IL-1 alpha, -1 beta, -3, -4, -5, -6, granulocyte-macrophage (GM)-CSF, TNF, and IFN-gamma. However, IL-2EF failed to enhance: 1) the activity of IL-1, -3, -4, -5, or -6 on cells responsive to these cytokines; 2) IL-2-augmented, IL-5-induced BCL1 proliferation; and 3) either PHA- or Con A-stimulated thymocyte proliferation. Moreover, neither IFN-gamma nor GM-CSF exhibited IL-2EF activity. When DC and T cells were cultured separately (after an initial 12 h co-culture period), IL-2EF activity resided predominantly in the T cell-derived supernatants. These and other data indicate that IL-2EF, a heat-labile T cell-derived 12.5 to 17.0 kDa protein, is distinct from IL-1 alpha, -1 beta, -2, -3, -4, -5, -6, TNF, IFN-gamma, GM-CSF, and previously described factors that co-stimulate thymocyte proliferation in the presence of Con A or PHA. It is suggested that IL-2EF functions to specifically enhance IL-2-driven T cell proliferation and contributes to the potent activation of T cells induced by DC.

VH gene expression by nontransformed pre-B cells upon differentiation in vitro.

Mice have more than 1000 VH gene segments, and each pre-B cell must choose a single one for rearrangement to encode the V portion of the antibody H chain. Presumably, all or most of the functional VH gene segments must be chosen by the population of B lymphocytes if the organism is to express the diversity that is observed in the immune system. Control of the selection of a VH gene segment for expression is not understood. We have found that the members of the VH gene family closest to the constant genes, the 7183 family, are transcribed in a manner that is specific for the stage of B cell development after pre-B cells derived from spleens of 6- to 8-wk-old nude mice are induced to differentiate in vitro by a mixture of dendritic cells and mitogen-activated T lymphocytes (DC-T). DC-T from spleens and lymph nodes induce transient high levels of synthesis of RNA from the 7183 VH family, whereas DC-T from Peyer's patches of mice of the same age as those from which spleen and lymph node DC-T were prepared did not induce the expression of RNA from that gene family. Spleen and Peyer's patch DC-T induce secretion of similar total amounts of antibody. Therefore, the RNA synthesis from members of at least one VH gene family is specific both for the lymphoid tissue in which B cell differentiation occurs and for the developmental stage of the B lymphoid cells.

Suppression of immune response induction in Peyer's patch lymphoid cells from mice infected with mouse hepatitis virus.

Multiple previous studies have demonstrated significant alterations of immunologic parameters associated with mouse hepatitis virus (MHV) infection, but effects of the virus on mucosal lymphoid cells have not been examined. Coincident with a natural outbreak of MHV at our institution, we noted alterations in immunoglobulin secretion by mature Peyer's patch B cells under an inductive stimulus provided by dendritic cells and mitogen-activated T cells (DC-T). MHV was isolated from mice affected during the outbreak, and experimental infection of mice with the isolate consistently resulted in failures of immunoglobulin secretion by cocultures of Peyer's patch DC-T and B cells. In subsequent experiments, MHV appeared to negatively affect DC-T more than B cells. Therefore, the effects of inapparent MHV infection on experimental mucosal immune responses can result from natural infection and can be experimentally reproduced.

A novel pre-B cell precursor: phenotypic characterization and differentiation induction by dendritic cell-T cell mixtures.

We have derived from spleens of nude mice early B lineage cell lines that are dependent upon factor(s) in supernatants of the WEHI-3B cell line. The cells are phenotypically at the pre-pre-B cell stage of differentiation. These cells are large and have basophilic granules in the cytoplasm. They are negative for cytoplasmic and surface immunoglobulin heavy or light chain, surface B220, surface Thy 1.2 and surface Ia but are positive for cytoplasmic B220. These cells at this early stage of maturation can be induced by mixtures of DC-T cells to express B220 on their surfaces and later to synthesize and ultimately to secrete immunoglobulin. These events are associated with morphologic changes in the cells to a lymphoblastoid appearance. Different pattern of immunoglobulin secretion were induced by DC-T from different tissues. The inductive event appears to occur during cell contact of pre-pre-B cells with the inducing DC-T cell mixture, but it does not appear to require IL-3. These data indicate that DC-T cell mixtures can provide signals for B cell differentiation at pre-pre-B cell stage as well as pre-B and mature B cell stage.

Massive hemorrhage from a lumbar artery following percutaneous renal biopsy.

We report a case of severe lumbar artery bleeding following percutaneous renal biopsy. A 68-year-old man with a history of rheumatoid arthritis, gold therapy, and Staphylococcus aureus bacteremia underwent a percutaneous renal biopsy to evaluate nephrotic syndrome and renal insufficiency. Following the procedure, he developed signs of severe hemorrhage. A selective renal angiogram revealed an intrarenal bleeding site that was occluded by selective embolization. The patient failed to stabilize however, and repeat angiography was performed two days later. A lumbar artery was identified as a second bleeding site, and was also occluded by selective embolization. The bleeding was controlled, but the patient developed serious complications and died five days later.

Different pathways of differentiation of pre-B cell lines are induced by dendritic cells and T cells from different lymphoid tissues.

Nontransformed pre-B cells were induced to differentiate in vitro along several different but predictable pathways with only dendritic cells (DC) and concanavalin A-stimulated T lymphocytes. DC-T from spleen induced secretion of only IgM, whereas DC-T from Peyer's patches induced high levels of IgA and intermediate levels of IgM and IgG. Both the isotype of antibody secreted and the extent of pre-B cell differentiation were determined by the lymphoid tissue source of DC, not of T cells. The pre-B cells synthesizing detectable levels of only the IgM heavy chain had cytoplasmic RNA transcripts from both light chain constant region genes and from the entire length of the heavy chain constant locus. In cells secreting IgM there were deletions in the DNA flanking the Cmu coding region.

Alterations in macrophage collagenase secretion induced by gold sodium thiomalate.

The effects of gold sodium thiomalate (GST) on the production of specific collagenase by thioglycolate-elicited macrophages was investigated. Our studies demonstrated that GST administration can significantly decrease collagenase production in a dose-dependent manner. These effects were observed with levels of GST attainable in serum or synovial tissue during routine chrysotherapy. In addition, GST altered lysozyme secretion by activated macrophages in a pattern distinct from that of collagenase alteration. These effects of enzyme secretion were not secondary effects of GST on viability, general protein secretion, or the specific assay procedures utilized, and were not attributable to the thiomalate moiety. Thus, GST may exert its therapeutic effect in rheumatoid arthritis through interference with the production of degradative proteolytic enzymes, which are important effector molecules mediating tissue destruction.

IgA rheumatoid factor synthesis by dissociated synovial cells. Characterization and relationship to IgM rheumatoid factor synthesis.

We examined patterns of IgA rheumatoid factor (RF) and IgM-RF synthesis by dissociated synovial cells obtained from 27 patients with seropositive rheumatoid arthritis. Synthesis of IgA-RF was observed in 19 of 34 synovial cell preparations from these patients and constituted a mean of 16% of the total IgA produced. IgA-RF expression correlated only weakly with IgM-RF production (r = 0.385) and could be dissociated from production of IgA-RF (and IgM-RF) exhibited by simultaneously obtained peripheral blood plasma cells. While wide variations were observed in the ratio of IgA-RF:IgM-RF produced by synovial B cells in the patient sample studied, remarkable consistency in the relationship of IgA-RF to IgM-RF synthesis was observed over time in different joints of the same patient. IgA-RF synthesized by dissociated synovial cells was predominantly of the IgA1 subclass and existed in both monomeric and polymeric forms. Our results are compatible with the view that local production of IgA-RF and IgM-RF are regulated independently of each other.

Continuing medical education. Changing behavior and improving outcomes.

A study was undertaken to determine if an intensive continuing medical education program in rheumatology could improve patient care. Fifteen primary care practitioners, who fit the description of educationally influential physicians, completed a 2-week academic medical center-based preceptorship. Improvement in physician knowledge, from a mean score of 65.3% to a mean of 82.9%, was documented using pre- and post-tests. Significant changes in physician behavior were documented using chart audits and patient interviews. The use of diagnostic tests and corticosteroids, and physician-patient interactions were the areas of greatest improvement. Functional outcomes for patients, measured by the Sickness Impact Profile, also improved. These findings suggest that a well-designed continuing medical education program can effect some changes in physician knowledge and behavior that will result in at least short-term improvement in patient outcomes.

The estrogenic activity of DDT: the in vitro induction of an estrogen-inducible protein by o,p'-DDT.

Induced protein is a commonly measured marker for estrogenic action. The induction of induced protein by o,p'-DDT was studied in an in vitro system. Nuclear levels of estrogen receptor translocated by o,p'-DDT correlated highly with induced protein induction, and the time course for induced protein induction was consistent with an estrogen receptor-mediated mechanism. While the maximum amount of induced protein produced by o,p'-DDT was less than after 17 beta-estradiol exposure, the induced protein formed by each compound was indistinguishable on nondenaturing and denaturing polyacrylamide gels. Also, it was shown that o,p'-DDT does not cause additional induction of induced protein over that seen with maximum levels of 17 beta-estradiol, further supporting the premise that these compounds share a common pathway in stimulating the synthesis of induced protein.

Preferential induction of polyclonal IgA secretion by murine Peyer's patch dendritic cell-T cell mixtures.

Polyclonal IgA secretion is inducible in murine B cells when DC-T from Peyer's patches (PP) provide the inducing stimulus. PP DC-T, which are composed predominantly of dendritic cells and Lyt-1+ T cells, are capable of dramatic augmentation of IgA secretion by PP or spleen B cells with minimal induction of IgM secretion. DC-T from spleen, however, are incapable of augmenting IgA secretion by either PP or spleen B cells. The level of IgA secretion is dependent upon the dose of DC-T providing the inducing stimulus and reaches a plateau with DC-T:B ratios of less than 1:1. This system for preferential induction of IgA responses should permit elucidation of cellular mechanisms involved in regulation of IgA secretion.

Mucosal immunoregulation: environmental lipopolysaccharide and GALT T lymphocytes regulate the IgA response.

In this review, we have emphasized: 1) bacterial lipopolysaccharide (LPS) involvement in IgA responses to orally administered thymic-dependent (TD) antigens; 2) characterization of Peyer's patch (PP) lymphoreticular cells; and 3) gastrointestinal immunization with gram negative pathogens and anti-LPS immunity to infection. Gut LPS, which interacts with PP lymphoreticular cells, is a major determinant for host responses to orally administered TD antigens. Bacteroides species are the principal microflora present in the gastrointestinal tract and our studies with phenol-water LPS extracts from Bacteroides fragilis indicate that both polysaccharide and lipid A activate lymphoreticular cells. The B. fragilis lipid A moiety, like that derived from E. coli and Salmonella LPS, induces B cell mitogenic responses in cultures from LPS responsive mice, but does not stimulate C3H/ H3J B cells. The inability of lipid A to stimulate gut-associated lymphoreticular tissue (GALT) cells of C3H/HeJ mice results in the induction of greater T helper cell activity in this tissue in response to orally administered TD antigens and ultimately results in an elevated IgA response pattern. Murine PP contain accessory cells (approximately 1% dendritic cells and 6-8% macrophages) and lymphocytes T (35-38%) and B (40-42%). Recent studies with antigen-specific T cell clones from C3H/ H3J PP have resulted in the isolation of IgA isotype-specific T helper cells (PP Th A cells). PP Th A cells are antigen-specific, bear Fc alpha receptors, and require H-2 histocompatibility with B cells for helper activity. PP Th A cells most effectively collaborate with surface IgA (sIgA)-bearing B cells (IgA committed B cells) for IgA isotype responses. Other studies have shown that PP dendritic cells and T cells form clusters when stimulated in vitro with sodium periodate and that these clusters promote polyclonal IgA responses in B cell cultures. Polyclonal IgA responses in cultures containing PP cell clusters from C3H/ H3J mice are considerably higher than those in identical cultures from LPS responsive mice. In other studies, the environmental influence on GALT B cells and their resultant commitment to IgA isotype is under investigation. CBA/N, X-linked immunodeficient (xid) mice possess an immature splenic B cell population which cannot respond to thymic-independent class-2 (TI-2) or certain TD antigens. However, GALT B cells of xid mice possess a mature Lyb-5+ B cell subpopulation capable of both TI-2 and TD responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Accessory cells in murine Peyer's patch. I. Identification and enrichment of a functional dendritic cell.

Previous studies have suggested that in vitro and in vivo immune responses are defective in Peyer's patch (PP) as a result of a deficiency in accessory cell number or function. However, we report here that enzymatic dissociation of PP does release a cell population with accessory activity in oxidative mitogenesis, i.e., the proliferation of periodate-modified T cells. The accessory activity present in PP is quantitatively similar to that of spleen. Accessory function is mediated by a cell type(s) that has the following characteristics: low buoyant density, lack of adherence to plastic or glass surfaces, lack of Fc receptors, and presence of surface Ia and the 33D1 dendritic cell (DC)-specific determinants. This PP accessory cell was markedly enriched by a novel technique. PP cells formed large aggregates when cultured for 16 h with irradiated, periodate-treated spleen cells. From the clusters we obtained a low density cell population that was 60% Ia positive, 33D1 positive, non-T and non-B, Fc receptor-negative, and dendritic in morphology. The DC-enriched populations were 60-80-fold enriched in accessory function relative to unfractionated PP. We can now compare PP accessory cells with accessory cells from other organs, and try to determine how PP dendritic cells contribute to the unique functions of this lymphoid organ.