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Biomedical subjects

R G Lynch

Publications and source records attributed to R G Lynch.

At least 19 recordsLinked to original sources
PubMed2000

A new non-radioactive method for IL-2 bioassay.

An oxidation-reduction (redox) indicator, alamarBlue, was used to measure the bioactivity of interleukin 2 (IL-2). This assay system has several advantages over other bioassays for measuring IL-2. It is a nonradioactive method unlike the conventional tritium-labeled thymidine ([3H]TdR) incorporation assay. The alamarBlue assay is also easier to use than other colorimetric methods, such as the MTT assay, because the alamarBlue assay does not depend on the extraction of insoluble formazan salt, which is time-consuming, error-prone, and cumbersome. Due to its solubility in culture medium and its nontoxicity to cells, alamarBlue provides an easy method to monitor cellular growth using either a fluorescence- or an absorbance-based instrument. The alamarBlue assay is not sample-destructive, unlike the thymidine incorporation and MTT methods. This adds another advantage to the alamarBlue method as the measurement of cellular growth by sample-destructive methods requires as many tubes as time points whereas the alamarBlue method requires only one tube for the entire growth period. In this study, alamarBlue was used to measure the proliferation of the IL-2-dependent cytotoxic T cell line, CTLL-2. The colorimetric change of alamarBlue at 570 nm compared to the reference wavelength, 600 nm, was proportional to the number of viable cells. The sensitivity of the IL-2 assay using alamarBlue was comparable to that of the [3H]thymidine incorporation method. These results demonstrate that the alamarBlue assay is valid for the IL-2 bioassay and that alamarBlue can replace the [3H]thymidine employed in the conventional proliferation assays.

Animals↗
PubMed2000

Regulatory roles for FcgammaRIII (CD16) and FcgammaRII (CD32) in the development of T- and B-lineage lymphoid cells.

IgG Fc receptors (FcgammaR) occur on all hematopoietic lineages and participate in a diversity of functions that reflect the combined effects of the molecular heterogeneity of FcgammaR and the inherent specialization of the FcgammaR+ cells. An extensive literature describes the functions of FcgammaR on mature myeloid and lymphoid cells in humans and mice but little has been published about FcgammaR on lineage progenitor cells. Several studies suggest that FcgammaR can influence leukocyte development and that FcgammaRII (CD32) and FcgammaRIII (CD16) can regulate murine T- and B-lineage development at stages before the expression of clonal antigen receptors. The nominal ligand of FcgammaR is IgG and the physiologically relevant ligand is the IgG-antigen complex, but it is also known that alternative, non-Ig ligands exist for Fc receptors. A role for FcgammaR in the regulation of leukocyte development has potential relevance for clinical situations in which the levels of nominal and/or alternative ligands of FcgammaR are elevated, or the production of soluble forms of FcgammaR is increased.

Animals↗
PubMed1999

Gammadelta T cell response induced by vaginal Herpes simplex 2 infection.

The purpose of the present studies was to determine whether acute vaginal infection with Herpes virus 2 altered the vaginal population of gammadelta T cells, and whether gammadelta T cells influenced the vaginal clearance of HSV-2. BALB/c mice were infected intravaginally with the progressively lethal wild type 333 strain, or the non-lethal thymidine kinase deficient (deltaTK- -HSV-2) mutant strain of HSV-2 virus. Changes in vaginal T cell composition were examined by FACS analysis 4 days after infection. Clearance of vaginal deltaTK- -HSV-2 infection was compared between mice with normal gammadelta T cell populations (BALB/c) and transgenic mice in which all the gammadelta T cells express a receptor that is specific for the b allotype of MHC class Ib T10 antigen (G8/BALB/c). In HSV-2 infected BALB/c mice, but not G8/BALB/c, a subset of gammadelta T cells that express a Vgamma2 TCR accumulated in the vaginal mucosa by the fourth day after infection. Unexpectedly, we found that gammadelta TCR transgenic mice exhibited a more rapid clearance of the virus than control mice (P < 0.05). These findings argue against the hypothesis that the normal populations of vaginal intraepithelial gammadelta T cells play a direct role in the elimination of virally infected epithelial cells.

Animals↗
PubMed1999

A regulatory role for Fc gamma receptors (CD16 and CD32) in hematopoiesis.

Progenitor cells of the T- and B-lineages in mice express (CD32) and Fc gamma RIII (CD16) but as the developing lymphocytes begin to express clonal antigen receptors, CD16 and CD32 are downregulated in T-cells, and CD16 is downregulated in B-cells. Considering that counter-receptors for Fc gamma R occur on thymic and bone marrow stromal cells, the possibility exists that Fc gamma R might participate in some aspect of T- and B-lineage development prior to the stage of antigen receptor expression. Previous studies provided evidence that Fc gamma R can influence murine T-lineage development. In the present studies we found that anti-Fc gamma RII/III mAb accelerated B-lineage development in bone marrow cultures from normal mice, but not in cultures from CD16-/- or CD32-/- mice. Similar results were observed when FACS-purified B-progenitor cells were co-cultured with BMS2, a bone marrow stromal cell line. Fresh bone marrow from CD32-/- mice contained about two-fold more B-lineage cells compared to bone marrow from normal or CD16-/- mice. These studies indicate that the Fc gamma R on B-lineage progenitor cells can influence their further development and add to a growing body of evidence that implicates Fc gamma R as regulatory elements in hematopoiesis.

Animals↗
PubMed1999

Characterization of novel FcepsilonRII/CD23 isoforms lacking the transmembrane (TM) segment in human cell lines.

Human FcepsilonRII/CD23 is an approximately 45 kDa type II transmembrane glycoprotein belonging to the C-type animal-lectin family, and has two isoforms (a and b) that only differ in their intracytoplasmic tails. We previously found that in several human and mouse cell lines there were two additional CD23 transcripts (a' and b') lacking the exon 3 that encodes the entire transmembrane segment and a part of cytoplasmic tails. In this study, we analyzed the putative CD23a' and CD23b' products at protein levels and characterized with rabbit polyclonal antibodies against novel amino-acid sequences of the putative CD23a' and CD23b' molecules (anti-CD23a' Ab, anti-CD23b' Ab). Western blots in COS cells transfected with CD23a' or CD23b' cDNA as well as in vitro translation assays showed that the a' and b' CD23 transcripts were translated to about 40 kDa molecules. These 40 kDa molecules were also recognized by a polyclonal antibody against 25 kDa soluble fragment of human CD23. We also found that human cells having mRNAs for CD23a' and CD23b' expressed protein products recognized specifically by anti-CD23a' or anti-CD23b' Ab, respectively. In addition, the CD23a' and CD23b' molecules in transfected COS cells were resistant to Endo H(f) and PNGase F, although these truncated forms as well as the membrane-associated forms had an asparagine residue responsible for the N-linked glycosylation. Taken together, our results show that the a' and b' CD23 transcripts are expressed and translated in human lymphoid cells and that their translated products are retained in the cytoplasm where they might play an unique regulatory role in the expression of the full-length CD23 on the cell surface.

Amino Acid Sequence↗
PubMed1998

A regulatory role for Fcgamma receptors CD16 and CD32 in the development of murine B cells.

Early in development, murine B-lineage progenitor cells express two classes of IgG Fc receptors (FcgammaR) designated as FcgammaRII (CD32) and FcgammaRIII (CD16), but mature B lymphocytes only express FcgammaRII (CD32), which functions as an inhibitor of B-cell activation when it is induced to associate with mIgM. The functions of CD16 and CD32 on B-lineage precursor cells have not previously been investigated. To search for FcgammaR functions on developing B-lineage cells, normal murine bone marrow cells were cultured in the presence of 2.4G2, a rat monoclonal antibody that binds to CD16 and CD32, or in the presence of control normal rat IgG, and then the B-lineage compartment was analyzed for effects. Cultures that contained 2.4G2 showed enhanced growth and differentiation of B-lineage cells compared with control cultures. The enhancing effect of 2.4G2 also occurred when fluorescence-activated cell-sorted B-cell precursors (B220(+), sIgM-, HSAhigh, FcgammaR+) from normal bone marrow were cocultured with BMS2, a bone marrow stromal cell line, but not when they were cultured in BMS2-conditioned media. The enhancement of B-lineage development induced by 2.4G2 was CD16-dependent and CD32-dependent, because 2.4G2 did not effect B-lineage growth or differentiation in cultures of bone marrow from mice in which either the gene encoding CD16 or CD32 had been disrupted. Analysis of fresh bone marrow from the CD16 gene-disrupted mice showed normal numbers and distribution of cells within the B-cell compartment, but in CD32 gene-disrupted mice, the B-cell compartment was significantly enlarged. These experiments provide several lines of evidence that the FcgammaR expressed on murine B-cell precursors can influence their growth and differentiation.

Animals↗
PubMed1998

Homing of transgenic gammadelta T cells into murine vaginal epithelium.

The vaginal epithelium of normal mice contains lymphocytes of fetal thymic origin that express an invariant Vgamma4/Vdelta1 TCR. The apparent lack of other gammadelta TCR species suggests that a selection mechanism might operate to regulate the localization of gammadelta T cells at this anatomical site. Selection might be connected to the Vgamma4/Vdelta1 TCR or to some homing characteristic of the fetal thymic lineage that appears at day 17-18 of embryonic life. In the present studies, we investigated whether transgenic gammadelta cells expressing a TCR species characteristic of the subpopulation of gammadelta T cells found in the blood, spleen and lymph would translocate to the vaginal epithelium. We found that the transgenic Vgamma2 TCR+ cells did accumulate in the vagina of transgenic mice. Furthermore, like normal vaginal gammadelta T cells, the transgenic vaginal gammadelta T cells expressed the phenotype of recently activated memory/effector T cells (CD44(hi), CD62L-, CD45RB(lo), CD69+). Vaginal gammadelta T cells in normal mice do not express the CD2 and CD28 antigens, but both of these markers are present on transgenic vaginal gammadelta T cells. We observed that a small fraction of splenic transgenic gammadelta T cells had the same surface phenotype as the vaginal transgenic gammadelta T cells, raising the possibility that the gammadelta T cells present in the vaginal epithelium of transgenic mice originated from the peripheral lymphoid organs. Data in support of this possibility came from experiments in which co-incubation of splenic transgenic gammadelta T cells with vaginal epithelial cell suspensions induced the vaginal gammadelta phenotype on the splenic gammadelta T cells. The finding of transgenic gammadelta T cells in the vaginal epithelium suggests that homing of gammadelta T cells to this site is not restricted to gammadelta T cells that express the V4/NS1 invariant TCR. Furthermore, these findings imply that retention of gammadelta T cells in the vaginal epithelium of normal mice is affected by a Vgamma4/Vdelta1-specific mechanism. The finding of a significant level of apoptosis in the transgenic vaginal gammadelta T cells, but not in the normal vaginal gammadelta T cells, could reflect that the mechanism of retention of Vgamma4/Vdelta1 + in the vaginal epithelium involves selective survival at the site.

Animals↗
PubMed1998

Rapid development of severe hyperplastic gastritis with gastric epithelial dedifferentiation in Helicobacter felis-infected IL-10(-/-) mice.

Interleukin (IL)-10 is a potent anti-inflammatory and immune-regulatory cytokine. Mice deficient in IL-10 production (IL-10(-/-)) develop a spontaneous inflammatory bowel disease, indicating that IL-10 is an important regulator of the mucosal immune response in vivo. To study the role of IL-10 in the host response to gastric Helicobacter infection, stomachs of IL-10(-/-) and wild-type mice were colonized with Helicobacter felis, as a model of human H. pylori infection. Within 4 weeks of H. felis infection, wild-type mice develop a mild, focal chronic gastritis. In contrast, H. felis-infected IL-10(-/-) mice develop a severe hyperplastic gastritis, characterized by a dense, predominantly mononuclear cell inflammation of the mucosa and submucosa and epithelial cell proliferation and dedifferentiation. Within 4 weeks of H. felis infection, there are striking alterations in the character of the gastric epithelium from IL-10(-/-) mice, including a profound loss of parietal and chief cells, focal de novo production of acidic mucins, and marked epithelial proliferation with disordered epithelial architecture. These findings indicate that, in the absence of IL-10, the inflammatory and immunological responses of the murine host to gastric colonization with Helicobacter is a rapidly evolving pathological process with features that mimic those associated with H. pylori infection in humans. H. felis-infected IL-10(-/-) mice may provide a model with which to investigate the cellular and molecular changes that stem from gastric infection with H. pylori.

Animals↗
PubMed1997

Fc gammaRII (CD32) is linked to apoptotic pathways in murine granulocyte precursors and mature eosinophils.

Murine granulocytes and precursors express low-affinity IgG Fc receptors (Fc gammaR). We investigated the effects of FcyR ligation on the development of eosinophils in cultures of normal murine bone marrow. Eosinophilopoiesis was induced by culture of bone marrow cells in the presence of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin-3 [IL-3], and IL-5). Addition to the cultures of 2.4G2, a rat monoclonal antibody (mAb) that reacts with Fc gammaRII (CD32) and Fc gammaRIII (CD16), induced granulocyte apoptosis within 24 hours. Granulocytes in cultures that contained 2.4G2 showed chromatin condensation, binding of Annexin-V, and fas induction, and by electron microscopy, apoptosis was most commonly observed in cells of the eosinophil lineage. Since murine granulocytes can express both Fc gammaRII (CD32) and Fc gammaRIII (CD16), we investigated the effect of 2.4G2 on cultures of bone marrow obtained from Fc gammaRIII (CD16) gene-disrupted mice and found that the apoptosis induced with 2.4G2 was CD16-independent. Studies with bone marrow cultures from B6MLR-lpr/lpr and C3H/HEJ-gld/gld mice established that the Fc gammaRII (CD32)-triggered apoptosis was fas-fasL-dependent. When mature eosinophils isolated from hepatic granulomas of Schistosoma mansoni-infected mice were cultured in cytokines in the presence of 2.4G2, the eosinophils underwent apoptosis within 24 hours. These findings identify a previously unknown linkage between Fc gammaR on eosinophils and fas-mediated apoptosis, a connection that could be relevant to mechanisms by which eosinophils mediate tissue injury and antibody-dependent cellular cytotoxicity reactions.

Animals↗
PubMed1997

Lack of Fc-epsilon receptors on murine eosinophils: implications for the functional significance of elevated IgE and eosinophils in parasitic infections.

Chronic infection with Schistosoma mansoni induces in humans and mice a Th2-dominant immune response in which eosinophils and IgE are conspicuously elevated. Human eosinophils express IgE receptors that participate in an IgE-dependent eosinophil-mediated ADCC reaction against Schistosomula larvae in vitro. To investigate the expression of IgE receptors on murine eosinophils, they were purified (>95% pure by Giemsa-stained cytospin preparations) from liver granulomas of Schistosoma-infected mice. Flow cytometric analysis showed the absence of the low-affinity IgE receptor Fc-epsilon RII (CD23) and Mac-2 and the absence of binding of murine IgE. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of granuloma eosinophil mRNA did not detect transcripts for Fc-epsilon RII or the alpha-chain of the high-affinity IgE receptor Fc-epsilon RI, but did detect transcripts that encode Mac-2 and the low-affinity IgG receptors Fc-gamma RIIb2, Fc-gamma RIII, and the FcR-associated gamma-chain. In vitro stimulation of granuloma eosinophils with interleukin-4 (IL-4) did not induce IgE binding, surface expression of Mac-2, or the transcription of Fc-epsilon receptors (Fc-epsilon RI, Fc-epsilon RII/CD23). To investigate normal murine eosinophils, we cultured normal mouse bone marrow cells with recombinant IL-3, recombinant IL-5, and recombinant granulocyte-macrophage colony-stimulating factor, conditions that promote eosinophil differentiation. Flow cytometric analysis of bone marrow-derived eosinophils failed to detect IgE binding or cell surface expression of Fc-epsilon RII and Mac-2, and RT-PCR analysis of fluorescence-activated cell sorted bone marrow-derived eosinophils failed to detect transcripts that encode Fc-epsilon RI or Fc-epsilon RII. These findings show that, in contrast to human eosinophils, murine eosinophils do not express cell surface receptors that bind IgE. However, because IgG receptors (Fc-gamma RIIb2, Fc-gamma RII) were present on eosinophils purified from granulomas, we investigated whether they might be involved in eosinophil activation. We found that an oxidative burst in eosinophils could be triggered through their IgG receptors.

Animals↗
PubMed1997

Gamma delta T cells of the murine vagina: T cell response in vivo in the absence of the expression of CD2 and CD28 molecules.

While little is known about their activation requirements and function, the intraepithelial T cells of the murine vagina express TCR complexes in which the antigen recognition components and the signaling components have unusual features. These vaginal T cells express an invariant V gamma 4/V delta 1 TCR and appear to be the only intraepithelial gamma delta T cells that exclusively use FcR gamma chains in their TCR complex. To further characterize the vaginal gamma delta T cells we isolated them from normal mice and from mice injected systemically with an activation-inducing dose of anti-TCR mAb. The isolated gamma delta T cells were examined by flow cytometry for their surface expression of a panel of adhesion, proteins, activation antigens and cellular interaction molecules (CD44, CD62L, CD45RB, LFA-1, CD2 and CD28). The patterns of expression observed indicate that the vaginal gamma delta T cells of normal mice show the phenotype of effector T cells. The adhesion/co-stimulatory molecules CD28 and CD2 were not detected on vaginal gamma delta T cells, an interesting finding since the absence of CD2 from other T cells has been suggested to result in anergy. However, vaginal gamma delta T cells are responsive to TCR-mediated signals since injection of normal mice with pan-anti-TCR antibody or stimulating anti-gamma delta TCR antibody resulted in an increase in cell number and increased expression of transferrin and IL-2 receptors. These results indicate that vaginal gamma delta T cells might utilize other co-stimulatory molecules, if any, in connection with TCR-induced activation and differentiation. While the physiological function of vaginal gamma delta T cells remains unknown, the expression of an invariant V gamma 4/V delta 1 TCR, their exclusive use of gamma chain homodimers in their TCR, and the absence of CD2 and CD28 co-stimulatory molecules are a novel combination of properties that suggests specialized functional properties. Although vaginal gamma delta T cells share some features in common with gamma delta T cells that reside in other epithelial tissues, such as skin and intestine, the present studies provide additional evidence that vaginal gamma delta T cells are a highly specialized and distinct T cell population.

Animals↗
PubMed1996

Local suppression of IFN-gamma in hepatic granulomas correlates with tissue-specific replication of Leishmania chagasi.

BALB/c mice are susceptible to infection with the visceralizing species of Leishmania, Leishmania chagasi. The parasite load initially rises in the liver and spontaneously subsides, whereas parasite multiplication begins later and remains lower in the spleen. To investigate whether this organ-specific multiplication of L. chagasi correlates with localized immune responses, we compared cytokine production by splenic vs hepatic immune cells. Livers from infected mice contained granulomas harboring intracellular L. chagasi amastigotes, whereas few amastigotes were present in the spleen. FACS analysis granuloma cells showed granuloma lymphocytes expressed a memory/effector phenotype. Granuloma cells cultured in vitro produced IL-10 and IL-6 but no detectable IFN-gamma, IL-4 or IL-5. In contrast, splenocytes from the same animals secreted IFN-gamma, IL-4, IL-6, and IL-10. T cells were depleted from granuloma cells by immune lysis, and the results indicated that IL-10 and IL-6 were derived at least in part from a non-T cell compartment. Paradoxically, FACS-purified Thy-1+ granuloma lymphocytes were able to produce IFN-gamma in the absence of other granuloma cells, suggesting IFN-gamma production might usually be inhibited by a granuloma-associated non-T cell element. Coculture of splenocytes with either granuloma cells or supernatants from granuloma cultures inhibited the usual splenocyte production of IFN-gamma and IL-4 but not IL-10. Thus, there may be a unique granuloma-associated suppressive factor accounting for the absence of IFN-gamma in hepatic granuloma cultures. It may be the absence of IFN-gamma in the liver and presence in the spleen that allows or inhibits parasite survival, respectively, in these different locations.

Animals↗
PubMed1996

Activation features of intraepithelial gamma delta T-cells of the murine vagina.

The epithelium of the murine vagina contains a resident population of gamma delta T-cells that expresses a homogenous Vgamma4/Vdelta1 TCR lacking N-region junctional diversity, implying that these T-cells recognize a very limited array of antigenic structures. The vaginal gamma delta T-cells express a pattern of surface markers characteristic of memory/effector T-cells that have previously been activated. Although vaginal gamma delta T-cells do not express the major costimulatory molecules CD28 and CD2, they do proliferate in response to a systemically delivered anti gamma delta TCR stimulus. Vaginal gamma delta T-cells contain mRNA that encodes the keratinocyte growth factor raising the possibility that these cells play a role in the repair of vaginal epithelium following injury. While the antigen recognized by the vaginal gamma delta TCR is unknown, a model is proposed which attempts to relate some of the unusual phenotypic features of vaginal gamma delta T-cells to the physiological injury and shedding of vaginal epithelium that occurs during the estrous cycle.

Animals↗
PubMed1996

Cytokine secretion and adhesion molecule expression by granuloma T lymphocytes in Mycobacterium avium infection.

Mice experimentally infected with Mycobacterium avium develop a chronic disease characterized by widespread noncaseating granulomas. In this report, we describe the phenotype and cytokine secretion profile of these granuloma-infiltrating effector T lymphocytes. In response to specific antigen, granuloma T cells and, to a lesser extent, spleen cells secrete interferon-gamma, but no interleukin-4 or -5. The importance of this Th1-like response to the host was demonstrated by the massively increased bacterial load and lethal disease in interferon-gamma knockout mice. One function of localized cytokine secretion is to recruit inflammatory T cells bearing surface adhesion molecules complementary to counter-receptors on vascular endothelial cells. Granuloma T cells express high levels of these pro-inflammatory adhesion molecules but have down-regulated their expression of L-selectin (CD62L). The expression of these adhesion molecules on granuloma-infiltrating T lymphocytes would alter the migration pathway of these cells and is likely to be important in facilitating the traffic of effector T cells to the granulomatous inflammatory site. In addition, T cells from Schistosoma mansoni granulomas express the same set of adhesion molecules, showing that this phenotype is not specifically dependent upon the Th1 pattern of cytokine secretion.

Animals↗
PubMed1995

Two waves of gamma delta T cells expressing different V delta genes are recruited into schistosome-induced liver granulomas.

Isolated granulomas provide a unique model to study T cells in the site of inflammation. Schistosoma mansoni-infected mice develop liver granulomas in response to schistosome egg deposition and the granulomas contain gamma delta T cells that appear to be activated (Pgp-1high and L-selectin low). Analysis of kinetics and TCR gene usage of granuloma gamma delta T cells revealed a limited TCR repertoire restricted to V gamma 1.1, V delta 4, and V delta 6 genes, suggesting that the occurrence of gamma delta T cells in the granuloma is influenced by TCR V gene usage. The V delta 4+, but not the V delta 6+, gamma delta T cells expressed CD69, a marker of recent activation. To determine if there was a preferred order of accumulation of the gamma delta T cells in granulomas, s.c. sponge grafts were implanted into schistosome-infected mice, schistosome eggs were injected into the grafts, and accumulated T cells were sequentially analyzed. The earliest gamma delta T cell immigrants expressed V delta 6 and later immigrants expressed V delta 4 V genes. Additional evidence for a role of TCR specificity in the accumulation of gamma delta T cells in granulomas is their absence from schistosome granulomas in TCR transgenic mice that express only a single MHC-specific gamma delta TCR. Finally, gamma delta T cell recruitment into the granulomas did not require beta 2-microglobulin, since gamma delta T cells were present in liver granulomas of beta 2-microglobulin gene-disrupted mice. The analysis of the influx of gamma delta T cells into schistosome-induced, liver granulomas and schistosome egg-containing sponges provides a model system to investigate the role, if any, of gamma delta T cells in schistosome infections.

Animals↗