David M. Spalding

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 Peyers 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.

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 Peyers patch (PP) lymphoreticular cells; and 3) gastrointestinal immunization with gram negative pathogens and anti‐LPS immunity to infection.

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

Abstract 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 Peyers 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 Peyers 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.

Inhibition of Fucosylation Reshapes Inflammatory Macrophages and Suppresses Type II Collagen–Induced Arthritis

Fucosylation catalyzed by fucosyltransferases (FUTs) is an important posttranslational modification involved in a variety of biologic processes. This study was undertaken to determine the roles of fucosylation in rheumatoid arthritis (RA) and to assess the efficacy of reestablishing immune homeostasis with the use of 2‐deoxy‐d‐galactose (2‐d‐gal), a fucosylation inhibitor.

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.

Death Receptor 5–Targeted Depletion of Interleukin‐23–Producing Macrophages, Th17, and Th1/17 Associated With Defective Tyrosine Phosphatase in Mice and Patients With Rheumatoid Arthritis

OBJECTIVE Bidirectional interactions between granulocyte-macrophage colony-stimulating factor-positive (GM-CSF+) T cells and interferon regulatory factor 5-positive (IRF-5+) macrophages play a major role in autoimmunity. In the absence of SH2 domain-containing phosphatase 1 (SHP-1), GM-CSF-stimulated cells are resistant to death receptor (DR)-mediated apoptosis. The objective of this study was to determine whether TRA-8, an anti-DR5 agonistic antibody, can eliminate inflammatory macrophages and CD4 T cells in the SHP-1-deficient condition. METHODS Ubiquitous Cre (Ubc.Cre) human/mouse-chimeric DR5-transgenic mice were crossed with viable SHP-1-defective motheaten (mev/mev) mice. TRA-8 was administered weekly for up to 4 weeks. The clinical scores, histopathologic severity, and macrophage and CD4 T cell phenotypes were evaluated. The role of TRA-8 in depleting inflammatory macrophages and CD4 T cells was also evaluated, using synovial fluid obtained from patients with rheumatoid arthritis (RA). RESULTS The levels of inflammatory macrophages (interleukin-23-positive [IL-23+] IRF-5+) and CD4 T cells (IL-17+ GM-CSF+) were elevated in mev/mev mice. In DR5-transgenic mev/mev mice, DR5 expression was up-regulated in these 2 cell populations. TRA-8 treatment depleted these cell populations and resulted in a significant reduction in inflammation and in the titers of autoantibodies. In synovial cells from patients with RA, the expression of IRF5 and DR5 was negatively correlated with the expression of PTPN6. TRA-8, but not TRAIL, suppressed RA inflammatory macrophages and Th17 cells under conditions in which the expression of SHP-1 is low. CONCLUSION In contrast to TRAIL, which lacks the capability to counteract the survival signal in the absence of SHP-1, TRA-8 eliminated both IRF-5+ IL-23+ M1 macrophages and pathogenic GM-CSF+ IL-17+ CD4 T cells in a SHP-1-independent manner. The results of the current study suggest that TRA-8 can deplete inflammatory cell populations that result from a hyperactive GM-CSF/IRF-5 axis.

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.

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 Peyers 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.

[8] Antiisotypic antibodies

Publisher Summary This chapter provides an overview of antiisotypic antibodies. Antibodies capable of specifically binding to all molecules of an immunoglobulin class have proven to be invaluable reagents for a variety of purposes. This chapter reviews principles of preparation, characterization and application of antibodies. Sera (or ascites) containing substantial concentrations of a homogeneous Ig protein of the desired isotype (example, myeloma and hybridoma) are particularly advantageous starting materials for isolation of human, murine, and rat Ig proteins. Purified Ig proteins from several species are also available commercially. Regardless of the source, the purity of the proteins to be utilized for immunization should be ascertained by disc gel electrophoresis and immunoelectrophoresis against antiserum directed against serum proteins of the appropriate species prior to use. Enrichment of antiisotypic antibodies from heterologous antisera (or ascites/culture supernatant in the case of monoclonal antibodies) is frequently desirable prior to utilization. For heterologous antisera, either isolation of the IgG fraction of the serum or affinity purification of the antiisotypic antibody is usually required. Monoclonal antiisotype antibodies can generally be isolated from ascites (or culture supernatant) using affinity purification techniques. The chapter further discusses affinity purification of antiisotypic antibodies.

IDENTIFICATION OF A NONADHERENT ACCESSORY CELL IN MURINE PEYER'S PATCHES

Peyer’s patches (PP) have been considered functionally incompetent because specific antibody-producing plasma cells are not identifiable in PP after oral or parenteral immunization, and in vitro models have demonstrated that PP are incapable of mounting specific antibody responses. An explanation forwarded for this functional inadequacy of PP has been a deficiency of functional accessory cells (AC) in Peyer’s patches. It has recently been demonstrated, however, that PP cells obtained by enzymatic dissociation (with Dispase) are fully capable of in vitro mitogenesis, polyclonally activated immunoglobulin production, and primary in vitro antibody responses. These studies have indicated that PP possess functional AC. We have begun to characterize the AC population of PP and contrast it with the AC population of spleen (Sp). We have used oxidative mitogenesis (using sodium periodate) as an assay for accessory-cell activity. This assay has previously been demonstrated to require accessory cells, and dendritic cells are known to be the most potent stimulating population in the spleen. In our oxidative mitogenesis assay, irradiated (1500 R) stimulator populations to be assessed are cocultured with periodate-modified Sp T-cells (obtained by nylon wool purification), and pulsed with tritiated thymidine (3HTdR) at 24 hours. Cultures are harvested 24 hours later, and 3HTdR incorporation is assessed. Using this assay, enzymatic (Dispase) dissociation of PP releases a cell population with at least 5to 7-fold more AC activity than obtained with conventional (mechanical) dissociation. By contrast, an insignificant increase in accessory-cell activity is seen when Sp is enzymatically dissociated. Four murine strains (CD1, CBA/J, BALB/c, and C3H/HeN) show comparable results, and in these strains PP AC activity approaches spleen on a per cell basis. Artifactual alterations of PP populations by enzyme treatment have been eliminated as the source of increased accessory-cell activity. Experiments have been performed with total, adherent, and nonadherent populations as stimulators and reveal that AC activity released from PP is attributable to a radioresistant cell that resides predominantly (>75%) in the nonadherent fraction, whereas in Sp 9 5 % of the activity is present in the initially adherent fraction. Persistently adherent Fc receptor-bearing cells (macrophages) have been isolated from Sp and PP and are inactive as stimulator cells in this assay. Accessory-cell activity of PP is enriched in the cell pellicle obtained from flotation on dense bovine serum albumin columns ( p 1.078). The activity, again resides in the nonadherent fraction. Experiments using C3H/HeN (H-Zk] mice have demonstrated that the AC activity of PP is due to an Ia-bearing cell, as cytotoxicity with anti-Iak and rabbit complement abolishes AC activity of PP populations. Thus PP contains abundant accessory-cell activity in a cell population that is radioresistant, predominantly nonadherent, low density, Ia bearing, and lacking Fc receptors. These characteristics suggest a relationship with the Sp dendritic cell (described by Steinman and Cohn), but the appearance only after enzymatic dissociation and the lack of