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M C Howard

Publications and source records attributed to M C Howard.

At least 37 records · Page 2Linked to original sources
PubMed1995

CD38-mediated ribosylation of proteins.

The lymphocyte cell-surface Ag CD38 catabolizes NAD to adenosine 5' diphosphoribose (ADPR) and cyclic ADPR (cADPR). We show here that the soluble extracellular domain of CD38 (sCD38) mediates ADP ribosylation of several proteins. This was demonstrated by mass spectrometric analyses which revealed the addition of mass in units of 541.1 Da to these proteins, presumably corresponding to the covalent attachment of one or more ADPR moieties. Separate experiments showed that the same proteins became specifically radiolabeled following incubation with [32P]NAD plus sCD38. Additionally, it is shown that sCD38 can autoribosylate. Moreover, sCD38-mediated protein ribosylation was found to occur specifically at cysteine residues, since it was effectively blocked by addition of L-cysteine but not by other amino acids, and CD38-mediated protein ribosylation could be reversed by the addition of HgCl2, which specifically cleaves thiol-glycosidic bonds. ADPR purified from the reaction of sCD38 with NAD could itself be covalently transferred to target proteins at rates similar to the sCD38-mediated reaction, indicating that the ribosylation proceeds via the generation of this reactive intermediate. In vitro mutagenesis of a catalytic Glu residue that is conserved in numerous ADP-ribosyl transferases revealed that this amino acid is also important for catalysis in CD38. These data suggest that CD38 has the potential to cause ribosylation of experimental proteins, and raises the possibility that its specific ribosylation of a currently unidentified lymphocyte protein may contribute to its array of immunoregulatory activities.

ADP-ribosyl Cyclase↗
PubMed1995

Signaling through murine CD38 is impaired in antigen receptor-unresponsive B cells.

CD38 is a 42-kDa membrane associated enzyme which converts NAD into cyclic ADP-ribose (cADPR), a Ca(2+)-mobilizing second messenger, and ADP-ribose (ADPR). Agonistic antibodies to murine CD38 deliver a potent growth co-stimulus to mature splenic B lymphocytes. In this report we demonstrate a striking relationship between CD38-mediated mitogenesis and the ability of surface IgM to promote B cell proliferation. Tolerized B lymphocytes obtained from a double-transgenic mouse model of B cell tolerance do not proliferate in response to antigen stimulation through the Ig receptor or to agonistic anti-CD38 antibodies. Similarly, B-1 cells isolated from the peritoneal cavity of normal mice, and splenic B cells isolated from newborn mice were also unresponsive to both anti-IgM and anti-CD38 stimulation. All of these CD38-unresponsive B cells expressed normal levels of cell surface CD38 and responded to numerous other stimuli. CD38 immunoprecipitated from these B cell populations was normal in size and effectively hydrolyzed NAD, suggesting that the defect in CD38 signaling likely occurs downstream of CD38 itself. Signaling through CD38 and IgM does not always have identical effects on B cells since anti-CD38 cannot deliver inhibitory growth or differentiation signals to normal B cells or immature B cell lines. Nevertheless, the correlative data with these multiple B cell models of unresponsiveness suggests that the signaling pathway utilized by CD38 and IgM intersect, possibly sharing at least one of the crucial components of the Ig receptor signaling cascade.

ADP-ribosyl Cyclase↗
PubMed1995

CD38 expression on mouse T cells: CD38 defines functionally distinct subsets of alpha beta TCR+CD4-CD8- thymocytes.

We have examined CD38 expression on mouse lymphocytes using the rat mAb NIM-R5 and demonstrate that CD38 expression is restricted to approximately 8% of thymocytes. Although CD38 is absent from the majority of CD4+CD8- and CD4-CD8+ T cells, we detected a strong correlation between CD38 expression and alpha beta+CD4-CD8- T cells in the thymus, with nearly 80% of alpha beta TCR+CD4-CD8- thymocytes being CD38+. Using heat stable antigen (HSA) and CD38, we divided alpha beta+CD4-CD8- thymocytes into four subsets: HSA+CD38-, HSA-CD38hi, HSA-CD38low and HSA-CD38-. Two established characteristics of alpha beta TCR+CD4-CD8- cells, bias towards V beta 8.2 TCR expression and high levels of IL-4 production, were used to establish a possible relationship between the above thymocyte subsets. Our present data show that the HSA+CD38- subset is not biased towards V beta 8.2 TCR expression whereas the HSA-CD38- subset does show this bias (approximately 47%). Neither of these subsets make IL-4 upon CD3 mediated stimulation. In contrast, the CD38+ subsets are heavily biased toward V beta 8.2 expression and produce large amounts of IL-4 upon stimulation, particularly the CD38low cells. Taken together, these data suggest that these four subsets represent various stages of a possible differentiation pathway for alpha beta TCR+CD4-CD8- cells, with the HSA+CD38- subset being the most immature while the HSA-CD38low subset is the most functionally mature. These characteristics support the view that alpha beta TCR+CD4-CD8- T cells represent an independent lineage with a distinct, but as yet obscure, role in immunity.

ADP-ribosyl Cyclase↗
PubMed1994

Monoclonal antibodies to murine CD40 define two distinct functional epitopes.

Two rat IgG2a antibodies which define distinct epitopes on murine CD40 have been generated. These antibodies specifically bind recombinant murine CD40 expressed on L cells, and the soluble extracellular domain of murine CD40 coated onto microtiter plates. Both antibodies bind B220+ but not B220 murine spleen cells, and immunoprecipitate a 45-kDa protein from the surface of purified murine splenic B cells. These antibodies exhibit separate functional properties, consistent with the notion that they define two distinct CD40 epitopes. One of the monoclonal antibodies (designated 1C10) directly induces a specific proliferative response from mature murine B cells, up-regulates several B cell surface antigens, and rescues immature B lymphoma cells from anti-IgM-induced growth arrest. The other monoclonal antibody (designated 4F11) exhibits none of these properties, but is capable of synergizing with suboptimal amounts of either anti-IgM antibodies or the 1C10 agonistic anti-CD40 antibody to produce an optimal proliferative response of purified small dense B cells. Furthermore, 4F11 antibody synergizes with suboptimal amounts of 1C10 antibody to rescue B lymphoma cells from anti-IgM-induced growth arrest. The 1C10 and 4F11 antibodies were unable to cross-block each other's binding to recombinant CD40 expressed in L cells, providing strong support for the notion that the antibodies recognize distinct epitopes on CD40. The potential implications of two functionally distinct CD40 epitopes are discussed.

Animals↗
PubMed1993

Protective effects of IL-4 and IL-10 against immune complex-induced lung injury.

Recombinant murine IL-4 and IL-10 have been used in two models of inflammatory lung injury in rats after intrapulmonary deposition of IgG or IgA immune complexes. These models have contrasting requirements for cytokines, phagocytic cells, and adhesion molecules. In these two models of lung injury, IL-4 and IL-10 were individually coinstilled into the airways with the IgG or IgA antibodies, whereas the Ag were injected intravenously. Injury was quantitated by increases in permeability (leakage of 125I-BSA) and by hemorrhage (extravasation of 51Cr-RBC). In the model of IgG immune complex-induced lung injury, IL-4 and IL-10 were each highly protective when given in nanogram amounts. These protective effects were dose dependent. IL-4 and IL-10 caused substantial reductions in lung content of myeloperoxidase and parallel reductions in neutrophil content in bronchoalveolar lavage (BAL) fluids. The protective effects of IL-4 and IL-10 were associated with profound reductions of TNF-alpha in the BAL fluids and complete inhibition in the up-regulation of pulmonary vascular ICAM-1. In the IgA immune complex model of lung injury IL-4 had no protective effects, whereas IL-10 was highly protective. These protective effects correlated with diminished retrieval of alveolar macrophages in BAL fluids. These data suggest that IL-4 and IL-10 have significant protective effects in lung inflammatory injury, presumably achieving these effects by various mechanisms.

Animals↗
PubMed1982

Antigen-induced B lymphocyte differentiation.

The analysis of B-lymphocyte activation has been a tantalizing prospect for both immunologists and cell biologists, as B cells have the unique characteristics of possessing a well-characterized receptor and displaying a clear inducible response to a specific antigen. As outlined in this review, we now know much about the cellular events which occur when mature B cell encounters antigen within the peripheral lymphoid organ of an immunocompetent host. Our understanding of the molecular events associated with these cellular processes is currently limited, but such information will undoubtedly ensue. The greatest challenges remaining in B-cell immunology are immunoregulation and an understanding of how the enormous B-cell specificity repertoire is generated. These challenges require greater insight into lymphocyte triggering events and the early events in B-cell maturation. This review has not restricted itself to the phenomena associated with antigen-triggered mature splenic B cells, but has attempted to include aspects of B-cell development which are often overlooked or disregarded. In particular, attention has been drawn to certain nonspecific antigen-induced B-cell events, and to antigen-initiated developmental processes which precede mature B-cell development. Additionally, the commitment of triggered mature B cells to effector cell production has been reconsidered in light of current attempts to propagate normal lymphocytes continuously and in a manner which avoids terminal differentiation. The availability of such lines, together with increasingly refined technology, will hopefully allow us to resolve the remaining mysteries in B-cell development.

Animals↗
PubMed1979

Hybridoma antibody immunoassays for the detection of parasitic infection: development of a model system using a larval cestode infection in mice.

A prototype immunodiagnostic assay has been developed using chronic infection with the larval cestode, Mesocestoides corti, as a model system in mice. The assay is highly sensitive, it appears to be absolutely specific for M. corti infection, and is based on the inhibition of binding (by sera from infected mice) of a radiolabelled anti-M. corti hybridoma antibody to a crude M. corti antigen extract. The hybridoma antibody binds to living M. corti larvae and is an IgG1 protein. In large scale experiments no false positives were detected and the only M. corti-infected mice not detected by the assay were hypothymic nude (nu/nu) mice. Only limited success has been achieved in attempts to convert the assay to one not requiring parasite antigen and based on the inhibition of binding of radiolabelled anti-parasite hybridoma antibody and a large pool of anti-idiotype antiserum. Monoclonal antibodies derived from anti-parasite antibody-secreting hybridoma cell lines will be of particular use in the development of new, highly specific, immunodiagnostic reagents for the detection of parasite infection, exposure and disease.

Animals↗
PubMed1978

Distribution of surface IgM and IgD on single cells from lymphocyte subpopulations.

The surface immunoglobulin heavy chains on individual spleen cells fractionated by velocity sedimentation were studied using fluorescent antisera. In adult mice, cells bearing both mu and delta chains were found in all fractions. While there was an increase in the proportion of cells bearing mu only in the medium to large cell fractions, the majority of cells bearing mu only were small lymphocytes. Results obtained using 3-week-old mice were basically similar, but showed both a marked decrease in small mu + delta + cells and a marked increase in small mu + delta - cells when compared with adult animals.

Animals↗
PubMed1978

B lymphocyte subpopulations separated by velocity sedimentation. II. Characterization of tolerance susceptibility.

Neonatal and adult splenic B lymphocyte subpopulations, separated by velocity sedimentation, were tested in an in vitro splenic focus assay for their susceptibility to tolerance induction with hapten-protein conjugates. At least two tolerizable B cell subsets have been defined in the neonatal spleen; one comprising the slowly sedimenting, small lymphocytes and the other comprising the very rapidly sedimenting, large lymphocytes. In addition, a rapidly sedimenting large B cell subset in the adult spleen was found to be highly susceptible to tolerance induction. It was suggested that the inability to detect this susceptibility in unfractionated adult spleen was due to the low proportion of these very large lymphocytes in the total spleen cell population. The tolerizable B cell subsets presently defined probably represent the least mature B lymphocytes detectable in the splenic focus assay.

Aging↗
PubMed1978

Antigen-initiated B lymphocyte differentiation. XIV. Nonspecific effects of antigen stimulation cause proliferation in the "pre-progenitor" subset of primary B cells.

The effect of specific and nonspecific stimuli on the cycle status of subsets of primary B lymphocytes was assessed by preinjecting donor CBA mice 1 to 2 days previously with various substances, and then incubating the isolated spleen cells with high specific activity 3H-TdR before assay. AFC-progenitor activity was assessed as a response to NIP-POL antigen, either by adoptive transfer to irradiated recipients or by cell culture. Previous studies showed these assays reflected the activity of different subsets of B cells, termed "pre-progenitors" (adoptive assay) and "direct progenitors" (culture assay). Most functional primary B cells, whether assayed in culture or by adoptive transfer, were not initially in rapid cell cycle in normal adult mice. However, nonspecific stimulation for 1 day caused NIP-specific adoptive transfer IgM AFC-progenitors to enter rapid cell cycle. This effect was independent of T cells and not related to the antigenicity of the stimulus: particulate peritoneal irritants were the most effective stimulants. In contrast to adoptive transfer results. AFC-progenitors assayed in cell culture were unaffected by nonspecific stimuli, but were activated into cell cycle by specific antigen.

Animals↗
PubMed1977

Surface immunoglobulin characteristics of B-lymphocyte developmental states and B-lymphocyte colony-forming cells.

B-lymphocyte colony-forming cells and various B-lymphocyte developmental states were examined for expression of surface immunoglobulin. B-lymphocyte colony-forming-cells appeared to possess a significantly greater amount of surface immunoglobulin than did newborn 'virgin' B lymphocytes. In terms of surface immunoglobulin content and physical property characteristics (established previously) B-lymphocyte colony-forming cells resembled a more mature B-lymphocyte subset. This subset, as characterized in previous studies, consists of mature virgin B cells that develop from the newborn virgin B cells via a process of nonspecific antigen-induced differentiation. An alternative possibility that B-lymphocyte colony-forming cells are memory B lymphocytes has not been eliminated by this study.

Animals↗
PubMed1977

Antigen-initiated B lymphocyte differentiation: non-specific stimulation changes the physical properties of virgin AFC-progenitors in neonatal mouse spleen.

While the virgin AFC-progenitors for an adoptive immune response in neonatal germ-free CBA mouse spleen are small, dense cells, the equivalent cells in the adult are a larger, lighter density population. The effects of injections of unrelated antigens on the physical properties of the AFC-progenitors in neonatal spleen were investigated to test the postulate that the physically distinct "virgin" AFC-progenitors in the adult arose by a process of non-specific activation. Spleen cells from 7-day-old germ-free CBA mice were separated by sedimentation at unit gravity or by density on continuous albumin gradients, and the fractions were tested for NIP-specific AFC-progenitor activity using an adoptive immune assay which gave a direct linear measure of B cell activity. If the donor neonatal animals were injected one day previously with POL or PPD, the NIP-specific AFC-progenitor activity shifted from the typical small, dense lymphocytes to larger, lighter cells. The physical properties of these stimulated AFC-progenitors resembled those of IgM AFC-progenitors in normal adult mice. These results experimentally confirm the theory that environmental stimuli induce a non-specific "activation" of a particular subset of "virgin" B cells.

Animals↗