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Biomedical subjects

R L Coffman

Publications and source records attributed to R L Coffman.

At least 145 records · Page 8Linked to original sources
PubMed1988

Interleukin 4 causes isotype switching to IgE in T cell-stimulated clonal B cell cultures.

Although it has been established that IL-4 enhances both IgG1 and IgE secretion in LPS-stimulated B cell cultures, these studies failed to determine whether IL-4 preferentially induces isotype switching or preferentially allows for the maturation of precommitted precursor cells. To distinguish between these possibilities, it is necessary to ascertain the effect of IL-4 on the isotypes secreted by individual precursor cells during clonal expansion. Therefore, clonal cultures of B cells stimulated with a Th2 helper cell line specific for rabbit Ig and rabbit anti-mouse IgM were established. The majority of B cells are capable of undergoing clonal expansion under these conditions. To vary the level of IL-4 present, either IL-4 or anti-IL-4 was added to cultures. In the presence of IL-4 there was an increase in the proportion of clones that secreted IgE and a decrease in the proportion of clones that secreted IgM. The addition of IL-4 to cultures also increased the amount of IgE secreted by individual clones. Thus, these experiments definitively prove that IL-4 causes specific heavy chain class switching to IgE in Th2-stimulated B cell cultures. In contrast, IL-4 does not affect the proportion of clones secreting IgG1, suggesting that other consequences of Th cell-B cell interactions play a role in the generation of an IgG1 response.

Animals↗
PubMed1988

The characterization of four monoclonal antibodies specific for mouse IL-5 and development of mouse and human IL-5 enzyme-linked immunosorbent.

Four rat mAb directed against mouse IL-5 have been characterized by their ability to remove and neutralize mouse IL-5 activity in various bioassays. All four mAb absorbed IL-5 activity from solution. Although all were able to neutralize mouse IL-5 bioactivity, two were significantly more effective. These two were also able to neutralize the activity of mouse IL-5 delivered to B cells during a cognate-linked interaction with a Th cell clone. A two-site sandwich ELISA specific for mouse IL-5 was developed by using pairs of mAb. The mouse IL-5 ELISA is capable of detecting natural or mouse rIL-5 in supernatants, crude bacterial lysates, and high concentrations of mouse serum, and has a detection limit of less than 20 pg. Two of these antibodies cross-reacted with and neutralized human rIL-5, and one of these was used for development of an ELISA for human IL-5.

Animals↗
PubMed1988

Increased autoantibody production by NZB/NZW B cells in response to IL-5.

We previously demonstrated that B cells from NZB/NZW but not nonautoimmune mice secrete high levels of autoantibodies in response to factor(s) derived from type 2 Th cell (Th2) clones. Supernatants from type 1 Th cell clones, which contain a different set of lymphokines, were not stimulatory. In the present experiments, we attempted to define the active Th2 factor(s) and to better understand the cellular basis for the hyperresponsiveness. In response to optimal concentrations of supernatant (Th2-Sup), B cells from 3-mo-old NZB/NZW mice produced up to 40-fold greater amounts of IgM anti-DNA compared with unstimulated B cells, whereas BALB/c B cells produced levels only slightly above background. Although Th2-Sup contained large amounts of IL-4, comparable concentrations of rIL-4 alone did not stimulate NZB/NZW B cells. Furthermore, a blocking anti-IL-4 mAb did not prevent Th2-Sup-stimulated autoantibody production. Th2-Sup was fractionated by HPLC, and the stimulatory factor(s) was found in fractions known to contain IL-5 (also known as B cell growth factor II). Indeed, a highly purified preparation of IL-5 reproduced the effects of Th2-Sup by stimulating NZB/NZW B cells to produce high levels of IgM anti-DNA antibodies while enhancing production by nonautoimmune cells only slightly. In limiting dilution studies, NZB/NZW compared with BALB/c spleens contained a three- to four-fold greater frequency of DNA-specific B cells that were responsive to IL-5. Together, the results suggest a potential role for IL-5 in the pathogenesis of NZB/NZW autoimmune disease.

Animals↗
PubMed1988

Increase of precursor frequency and clonal size of murine IgE-secreting cells by IL-4.

IL-4 is able to preferentially enhance murine IgE levels in the supernatant of LPS-stimulated T cell-depleted splenic B cell cultures. Clonal and quantitative analysis of this response revealed that this is due partly to a 14-fold increased IgE precursor frequency and partly to a three-fold increased clone size of IgE-secreting cells. IL-4 increased the precursor frequency and the clone size of IgM-secreting cells not more than twofold. Both the IgM and IgE response in LPS-stimulated B cells were completely inhibited by the addition of anti-IgM mAb (M41) to the cultures, indicating that the IgE-secreting clones developed as subclones from precursors that express IgM. These cells lacked expression of membrane-bound IgE up to day 5 of the culture. Application of feeder cells in these cultures resulted in an increased precursor frequency of IgE-secreting clones among LPS-reactive B cells that is due, partially, to IL-4 produced by the feeder cells.

Adjuvants, Immunologic↗
PubMed1988

Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells.

The regulation of the subclass of immunoglobulin secreted by B cells has been studied in vitro in polyclonal systems using mitogens, such as lipopolysaccharide (LPS), to bypass the requirement for cognate interaction between antigen-specific T and B cells. In these systems, interleukin-(IL)-4 induces the secretion of IgG1 (ref. 1) and IgE (ref. 2); IL-5 enhances the secretion of IgA, and interferon-gamma (IFN-gamma) enhances the secretion of IgG2a (ref. 5). Clones of murine TH cells can be divided into two subsets, TH1 and TH2 (ref. 6). Both subsets synthesize IL-3 and granulocyte-monocyte colony-stimulating factor (GM-CSF), but only TH1 clones produce IL-2, IFN-gamma, and lymphotoxin (LT) and TH2 clones produce IL-4 and IL-5 (ref. 7). We have examined the role of clones of antigen-specific TH1 and TH2 cells in the regulation of the subclasses of IgG antibody secreted by antigen-specific B cells. Our results show that both types of TH cells induce the secretion of IgM and IgG3, whereas clones of TH1 and TH2 cells specifically induce antigen-specific B cells to secrete IgG2a and IgG1, respectively. We also demonstrate that regulation of commitment to the secretion of a particular IgG isotype occurs in two distinct stages: cognate interaction between T and B cells and interaction between T-cell-derived lymphokines and B cells.

Animals↗
PubMed1988

Identification of IL-6 as a T cell-derived factor that enhances the proliferative response of thymocytes to IL-4 and phorbol myristate acetate.

Thymocytes undergo a vigorous proliferative response when stimulated with a combination of IL-4 and PMA. We have found that conA-induced supernatants from a number of Th cell clones could enhance the level of IL-4/PMA-induced proliferation of unseparated thymocytes 0.5- to 2-fold and of peanut agglutinin-positive thymocytes 2- to 10-fold. These supernatants did not contain IL-2 or IFN-gamma, and the enhancing activity could be chromatographically separated from IL-3, -4, -5, and granulocyte/macrophage CSF. The possibility that the thymocyte enhancement factor contained in these supernatants was IL-6 was suggested when murine rIL-6 was found to have similar activity. Further evidence for the identity of these two factors was obtained when an IL-6 assay, based on plasmacytoma growth, was used to test column fractions showing thymocyte enhancement. All fractions active in the thymocyte enhancement assay also had activity in the plasmacytoma growth assay. These observations suggest that the thymocyte-stimulating activity present in the T cell supernatants was due to IL-6.

Adjuvants, Immunologic↗
PubMed1988

IFN-gamma regulates the isotypes of Ig secreted during in vivo humoral immune responses.

The lymphokine IFN-gamma has been shown in vitro to stimulate IgG2a secretion and inhibit IgG1 and IgE secretion by LPS-activated B lymphocytes. To determine whether IFN-gamma has a similar isotype regulatory role in vivo, we studied the abilities of rIFN-gamma and a mAb to IFN-gamma to modify the isotypes of Ig secreted in mice injected with a goat antibody to mouse IgD, which by itself induces large increases in levels of serum IgG1 and IgE and a relatively small increase in serum IgG2a. Multiple injections of IFN-gamma substantially inhibited production of IgG1 and IgE, and stimulated production of IgG2a in affinity purified goat antibody specific for mouse IgD-treated mice; anti-IFN-gamma antibody blocked the effects of IFN-gamma and in fact enhanced IgG1 and IgE secretion and inhibited the IgG2a response in these mice. The role of IFN-gamma in the selection of isotypes of Ig produced in response to injection of mice with the bacterium Brucella abortus (BA) was also studied, because killed, fixed BA are known to stimulate IFN secretion and a predominantly IgG2a antibody response. Anti-IFN-gamma antibody strongly suppressed IgG2a secretion and stimulated IgG1, but not IgE, secretion in BA-immunized mice. BA suppressed IgG1 and IgE secretion and enhanced IgG2a secretion in affinity purified goat antibody specific for mouse IgD-injected mice; treatment of these mice with anti-IFN-gamma antibody reversed the effects of BA on IgG1 and IgG2a secretion, but not the suppressive effect of BA on IgE secretion. These observations demonstrate that IFN-gamma has an important and perhaps unique physiologic role in the stimulation of IgG2a secretion and in the suppression of secretion of IgG1, whereas bacterial antigens can suppress IgE secretion by other mechanisms in addition to IFN-gamma secretion.

Animals↗
PubMed1988

Frequency analysis of functional Ig C epsilon gene expression in the presence and absence of interleukin 4 in lipopolysaccharide-reactive murine B cells from high and low IgE responder strains.

Nonresponder SJL mice produce low levels of antigen-specific IgE after immunization, compared to responder strains. Young athymic BALB/c nude mice are unable to produce antigen-specific or total IgE in their serum. These mice also have very numbers of background IgE-secreting cells in their lymphoid organs. High-responder BALB/c mice do have substantial numbers of background IgE-secreting cells while low-responder AKR mice show intermediate numbers. Similar differences were found when analyzing lipopolysaccharide (LPS)-reactive B cells in cell suspensions of spleen and bone marrow in limiting dilution cultures. Limiting dilution analysis of T cell-depleted splenic B cell cultures revealed that the defective IgE production in SJL mice is not due to an intrinsic B cell defect. This defect can be substantially overcome by addition of exogenous interleukin 4 (IL4) to these cultures. Furthermore, it was shown in limiting dilution cultures that SJL thymocyte feeder cells were able to suppress IgE production by LPS-activated high-responder BALB/c B cells. The addition of IL4 or neutralizing antibodies against IL4 or interferon-gamma to these cultures helped to overcome this suppressive effect to a large extent. We conclude that different IgE responder types are caused, at least in part, by a defective IL4 production or by a defect in the TH2 system that is functionally detectable at the level of thymocytes.

Animals↗
PubMed1988

Enhanced response of autoantibody-secreting B cells from young NZB/NZW mice to T-cell-derived differentiation signals.

Spleen cells from young NZB/NZW mice spontaneously produce IgM antihistone and anti-DNA antibodies in culture, and this in vitro autoantibody production is T-cell dependent. In the present studies, we investigated the response of young autoantibody-producing NZB/NZW B cells to various T-cell-derived signals. Stimulation with unprimed allogeneic T cells resulted in a 10- to 20-fold increase in IgM antihistone and anti-DNA antibody production compared with cultures of B cells alone. The responding cells were found in the large B-cell fraction after separation on Percoll gradients. Allo-stimulated B cells from nonautoimmune mice produced much lower absolute amounts of IgM autoantibodies as well as total IgM compared with NZB/NZW cells. Marked IgM antinuclear antibody and total IgM production was also observed when NZB/NZW B cells were cultured with supernatants from TH2 but not TH1 T-helper clones. Although B cells from nonautoimmune mice produced high levels of autoantibodies after stimulation with lipopolysaccharide, only minimal levels were secreted in response to the active supernatants. These results suggest that young NZB/NZW mice have IgM autoantibody-producing B cells that are more sensitive to certain T-cell-derived signals compared with B cells from normal mice. Although these hyperresponsive NZB/NZW cells appear to be in an advanced stage of activation, they require additional T-cell signals to express this abnormality.

Age Factors↗
PubMed1988

Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells.

Interleukin 6 (IL-6) refers to the gene product that was characterized initially as beta 2 interferon/26-kDa protein produced by human fibroblasts and later was found to be identical to B-cell stimulatory factor 2, hybridoma/plasmacytoma growth factor, and probably hepatocyte-stimulating factor. Using the human IL-6 cDNA as a probe, we have isolated functional cDNA clones from mouse bone marrow stromal cell cDNA libraries. Sequence analysis of the mouse cDNA insert revealed significant homology between the human and mouse IL-6 cDNA clones both at the level of nucleotide (65%) and deduced amino acid (41%) sequences. The NH2-terminal sequence of the deduced protein is identical to a partial NH2-terminal sequence determined previously for a hybridoma/plasmacytoma growth factor and a plasmacytoma growth factor isolated from mouse T cells and macrophages, respectively. The mRNA for mouse IL-6 is expressed in IL-1-treated stromal cells and in activated T-cell and macrophage cell lines. Supernatants from COS-7 monkey cells transfected with the cDNA clone have plasmacytoma growth factor, hepatocyte-stimulating factor, and colony-stimulating factor activities, as well as the ability to support the growth of a factor-dependent myeloid cell line, thus revealing an additional biological activity for IL-6.

Amino Acid Sequence↗
PubMed1988

A single antigen-specific B cell can conjugate to either a type 1 or a type 2 helper T cell.

Keyhole-limpet-hemocyanin-specific I-Ad-restricted T helper cells type 1 (TH1 cells) and type 2 (TH2 cells) were studied for their ability to physically conjugate to trinitrophenyl-specific antigen-binding B cells (TNP-ABCs). The same TNP-ABCs (which had already processed TNP-keyhole limpet hemocyanin) formed conjugates with one or the other T helper (TH) cell type, and conjugation was antigen-specific and major histocompatibility complex-restricted. The conjugation of a fixed number of one type of TH cell to the TNP-ABCs was inhibited by co-incubation with increasing numbers of cells of the other TH cell type. Thus, the vast majority of TNP-ABCs can conjugate to either type of TH cell. A similar pattern of inhibition of the conjugation of one TH cell type to the TNP-ABCs was seen when cells were co-incubated with increasing numbers of cells from an alloreactive T-cell clone. In all cases, conjugates contained only one T cell bound to one B cell, suggesting that interaction of an antigen-presenting B cell with one TH cell prevents the simultaneous binding of another TH cell.

Animals↗
PubMed1987

A mouse T cell product that preferentially enhances IgA production. I. Biologic characterization.

Supernatants from a subset of helper T cell clones can enhance IgA, IgE, and IgG1 production in cultures of lipopolysaccharide-stimulated, T cell-depleted spleen cells. The lymphokine interleukin (IL)-4 has been shown to cause the IgE and IgG1 enhancement produced by these supernatants. IgA enhancement, however, is mediated by a factor distinct from IL-4, although IL-4 can potentiate the effect of the IgA-enhancing factor. IgA-enhancing factor is also distinct from IL-1, IL-2, IL-3, granulocyte-macrophage colony-stimulating factor, and interferon-gamma and acts directly on B cells. Purified IgA-enhancing factor enhances IgA production three- to sixfold yet causes less than a twofold increase in other isotypes. The IgA enhancing activity is not inhibited by concentrations of interferon-gamma that inhibit IL-4 activities. In the accompanying article, we show that this IgA enhancing activity is a novel property of the lymphokine IL-5.

Animals↗
PubMed1987

A mouse T cell product that preferentially enhances IgA production. II. Physicochemical characterization.

Certain subsets of helper T cells, following stimulation with concanavalin A, secrete factors that specifically enhance the production of IgG1, IgE, and IgA by lipopolysaccharide-stimulated B cells. In the previous report, we describe a factor from the helper T cell line MB2-1 which enhances IgA production. IgA-enhancing factor has been purified from serum-free supernatants of this cell line. The purified lymphokine is a family of microheterogeneous polypeptides presumably modified post-translationally. IgA-enhancing factor has a native m.w. of 45,000 to 60,000 with subunits of between 24,000 and 28,000 under reducing conditions. Upon Edman degradation, a single amino-terminal sequence is detected which is identical to that of the lymphokine interleukin 5. IgA-enhancing factor activity is thus mediated by the same polypeptide that has been characterized as type II B cell growth factor, T cell-replacing factor, and eosinophil-differentiation factor.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of lymphokine cDNA clones encoding mouse and human IgA-enhancing factor and eosinophil colony-stimulating factor activities: relationship to interleukin 5.

Conditioned medium from the Con A-treated mouse helper T-cell clone Ly1+2-/9 contains activities that enhance the production of IgA by mouse B cells and induce human cord blood cells to form eosinophil colonies. We have isolated a cDNA sequence that expresses IgA-enhancing factor and eosinophil colony-stimulating factor activities from a cDNA library prepared from activated Ly1+2-/9 cells. Based on homology with the mouse cDNA sequence, a human cDNA sequence coding for an interleukin with IgA-enhancing factor and eosinophil colony-stimulating factor activities was isolated from a cDNA library prepared from a human T-cell clone stimulated with anti-T3 antibody and phorbol 12-myristate 13-acetate. DNA sequence analyses revealed that mouse and human cDNA clones encode proteins of 133 and 134 amino acids, respectively, that are identical to cDNA clones encoding the T-cell replacing factor I and B-cell growth factor II activities. These results establish that a single cDNA clone encodes a protein that acts as a growth and differentiation factor for both B cells and eosinophils.

Amino Acid Sequence↗