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Biomedical subjects

A Coutinho

Publications and source records attributed to A Coutinho.

At least 37 records · Page 2Linked to original sources
PubMed1998

Administration to mouse of endotoxin from gram-negative bacteria leads to activation and apoptosis of T lymphocytes.

Lipopolysaccharide (LPS) from gramnegative bacteria is a well-known T cell-independent B lymphocyte mitogen and macrophage/monocyte activator. While the conventional view holds that LPS is ignored by T cells, we report here that administration of LPS to mice activates all B cells, but also engages most CD4 and CD8 T cells, as measured by the expression of the activation markers CD69 and CD25 and by size increase. T cells recruited in endotoxin-treated mice showed, following in vitro stimulation by concanavalin A, altered patterns of cytokine production. In vivo, massive T cell apoptosis was evidenced in the days following LPS exposure. The present observation may contribute novel insights into the mechanisms of endotoxin shock and of the immunological consequences of gram-negative infections.

Animals↗
PubMed1998

Genetic control of natural antibody repertoires: I. IgH, MHC and TCR beta loci.

Global analysis of natural antibody repertoires has revealed a marked conservation of reactivity patterns within inbred mouse strains, and characteristic strain-specific differences. We have now analyzed the genetic control of reactivity repertoires, aiming at identifying the respective selection mechanisms. Multiparametric statistics of a large number of serum antibody reactivities scored by quantitative Western blot analyses using extracts from homologous tissues and bacteria readily distinguish the reactivity patterns of C57BL/6 and BALB/c, revealing homogeneity among genetically identical individuals. Antibody repertoires in the prototype strains can also be segregated from those expressed by the respective IgH congenics, BC.8 and CB.20, demonstrating that IgH-linked genes contribute to determining natural antibody repertoires. Conversely, strains sharing IgH haplotype also express distinct reactivity patterns, indicating that other genes participate in the selection of serum IgM repertoires. Two such non-IgH loci were now identified. Thus, analysis of four MHC-congenic strains demonstrated that MHC-linked control of natural antibody repertoires is likely to operate through differential selection of T cell repertoires, since (1) mice that are congenic at the TCR beta locus, and (2) BALB/c nude mice grafted at birth with pure thymic epithelium from either C57BL/6 or BALB/c also differ in their natural antibody repertoires.

Animals↗
PubMed1998

Functional diversity and clonal frequencies of reactivity in the available antibody repertoire.

The present experiments address functional antibody diversity and clonal distribution in murine available repertoires. IgM-containing supernatants were prepared by unbiased, polyclonal stimulation of resting splenic B cells from C57BL/6 mice, to ensure similar numbers of responding clones/culture and equivalent growth and maturation of all clones. The repertoires of clones and clonal mixtures were quantitatively assayed by limiting dilution analysis (LDA) on immunoblots of sodium dodecylsulfate polyacrylamide gel electrophoresis of homologous liver extracts, allowing to determine specific clonal frequencies towards the many hundred blotted antigens. The clonal frequency of reactivity of B cells with the extract was shown to be a bi-modal distribution of specific frequencies between 1/220 and 1/100,000. Cross-correlation analysis of reactivity to different bands in individual supernatants revealed low levels of cross-reactivity, suggesting that the blotted extract provides a very diverse set of antigens. Investigation of the affinity/concentration thresholds for detection of antigen-antibody interactions of our assay supports the notion that global repertoire analyses on immunoblots were highly discriminative and non-degenerate. Furthermore, reactivity patterns obtained with complex antibody mixtures correlated with the frequency of clonal reactivities as determined by LDA. The results demonstrate a large functional diversity of resting B lymphocytes, indicating a minimal repertoire size that is orders of magnitude higher than previous theoretical proposals suggested, and extensively heterogeneous in the size of clonal specificities.

Animals↗
PubMed1998

Naturally activated CD4+ T cells are highly enriched for cytokine-producing cells.

Most T cells in a normal non-immunized individual are in a resting state. However, a small proportion of splenic T cells are large activated cells both in specific pathogen-free and antigen-free mice. To further elucidate the effector functions associated with these "naturally" activated CD4+ T cells, we have characterized the expression of various membrane markers, cytokine production and T helper activity by these cells. We show that naturally activated CD4+ T cells express activation markers and contain tenfold higher proportions of cells producing IL-4, IL-10 and IFN-gamma as compared to small CD4+ T cells. Despite the high proportion of IFN-gamma producers, naturally activated CD4+ T cells still induce B cell proliferation and differentiation. These results are discussed in the context of normal physiological autoreactivity.

Animals↗
PubMed1998

Dynamics of serum IgM autoreactive repertoires following immunization: strain specificity, inheritance and association with autoimmune disease susceptibility.

Immunization of Lewis rats with myelin basic protein (MBP) in complete Freud's adjuvant (CFA) provokes experimental allergic encephalomyelitis (EAE). Here we compare, irrespective of antigen specificity, the structure and dynamics of serum IgM autoreactive repertoires following immunization with MBP/CFA in EAE-susceptible Lewis and relatively resistant Fischer rats. Prior to the appearance of clinical symptoms, Lewis rats developed a specific modification of serum IgM autoreactivities that, scored on other determinants than MBP itself, showed a prognostic association with EAE symptoms. Although comparable in their production of MBP-specific serum IgM and IgG antibodies, Fischer rats did not share these MBP/CFA-induced IgM autoreactivities of Lewis rats when immunized in the same manner. Moreover, while the Lewis-type repertoire reaction was specific for MBP/CFA alone, the respective Fischer reaction was not qualitatively different from that observed in this strain upon non-pathogenic immunization with self-related or -unrelated antigens. In general, the repertoire reactions differed qualitatively between the strains, consisting of components with typical behavior and strain preferences. The EAE-associated, as well as the other components of both Lewis- and Fischer-type repertoire reactions were usually co-dominantly inherited in F1 animals. These results indicate that a global antibody repertoire analysis may serve as a tool to describe prototypical response structures, possibly involved in immune regulation and susceptibility to pathogenic autoimmunity.

Animals↗
PubMed1997

Abnormal T cell selection on nod thymic epithelium is sufficient to induce autoimmune manifestations in C57BL/6 athymic nude mice.

To investigate the role of primary T cell repertoire selection in the immunopathogenesis of autoimmune diseases, pure thymic epithelium (TE) from nonobese diabetic (NOD) embryos was grafted into non autoimmune prone newborn C57BL/6 athymic mice. The results show that NOD TE selects host T cell repertoires that establish autoimmunity in otherwise nondiabetic animals. Thus, such chimeras regularly show CD4 and CD8 T cell-mediated insulitis and sialitis, in contrast with syngeneic or allogeneic chimeras produced with TE from nonautoimmune strains. This is the first demonstration that autoimmunity to pancreatic beta cells and salivary glands can be established by the sole alteration of the thymic environment involved in T cell selection, regardless of the nature and presentation of both major histocompatibility complex and tissue-specific antigens on the target organ. These data indicate that T cell repertoire selection by the NOD thymic epithelium is sufficient to induce specific autoimmune characteristics in the context of an otherwise normal host.

Animals↗
PubMed1997

Targeted disruption of the V(H) 81X gene: influence on the B cell repertoire.

We have generated a mutant mouse in which the most D-proximal V(H) gene (V(H)81X) has been disrupted by introducing a neomycin-resistance gene into the V(H)81X exon by means of gene targeting in embryonic stem cells. The mutant mice generated are unable to express the V(H)81X gene but appear to display a normal pattern of B cell differentiation as well as normal numbers of bone marrow and peripheral B cells from fetal life all through ontogeny. They mount normal immune responses to several different antigens tested. In contrast, the distribution of V(H) gene rearrangements in the V(H)7183 family is altered in homozygous mutant mice. Thus, the antibody repertoire of the targeted mice is modified, at least as far as the expression of V(H)7183 genes is concerned.

Animals↗
PubMed1997

Maternal IgG stimulates B lineage cell development in the progeny.

To examine the physiological role of maternal natural IgG antibodies on the development of B lineage cells of the progeny, we have bred homozygous muMT/muMT or heterozygous muMT/+ females to muMT/muMT or muMT/+ males, respectively. We could thus compare normal or B cell-deficient mice born from Ig-deprived (Ig-) or phenotypically normal mothers (Ig+). B cell-deficient progeny of heterozygous mothers contain no detectable serum IgA or IgM, but IgG concentrations that peak at 2 mg/ml by 7-21 days of age, decay after weaning with a half-life of 7 days, and remain detectable for 2 months after birth. At 7 days after birth, muMT/+ progeny born of Ig+ mothers contain two- to threefold higher numbers of bone marrow (BM) pre-B and B cells, and of splenic B cells, compared to mice of the same age born from Ig mothers. In contrast, the former progeny exhibit two to four times lower numbers of Ig-secreting plasma cells in spleen and thymus, and contain sixfold lower serum IgM concentrations. A similar maternal IgG-dependent stimulation of BM B cell precursors is also observed in muMT/muMT progeny. No significant differences were detected between the groups on day 3 after birth, suggesting the requirement for a minimal IgG concentration in the serum.

Animals↗
PubMed1997

The repertoire of serum IgM in normal mice is largely independent of external antigenic contact.

Antigen-free (AGF) and germ-free (GF) mice, although essentially free of serum IgG, maintain normal levels of circulating IgM. Using a quantitative immunoblot assay, we have now analyzed the repertoire of serum IgM from AGF, GF, and specific pathogen-free (SPF) BALB/c mice, on large panels of natural antigens from homologous tissues and bacteria. The reactivity profiles were very similar in the three groups of mice. Multiparametric statistic evaluation of the data showed that BALB/c animals, SPF, GF, and AGF mice constitute an homogeneous group with similar immunoreactivity profiles when compared to C57BL/6. Differences between immunoreactivity profiles of GF and AGF mice were observed, but were not statistically significant. These results suggest that the serum IgM repertoire of normal mice is strictly regulated and selected by endogenous ligands.

Animals↗
PubMed1997

An example of idiotypic mimicry.

We have evaluated the impact of transgenic immunoglobulin (TGIg) expression on endogenous antibody repertoires. The transgenic system was chosen as to allow for normal recombination of endogenous Ig genes, secretion of TGIg from early development on, and distinguishing the TGIg from endogenous Ig by several serological markers on the C and V regions of the molecules. The transgenic construct encodes a complete anti-(4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NP) antibody molecule carrying a well-defined idiotype, bearing a lambda 1 light chain and a chimeric heavy chain encoded by a human alpha 2 C region devoid of its membrane exon, and the murine B1.8 VDJ-region. Endogenous antibody repertoires were analyzed in mitogen-driven limiting dilution cultures, in single-cell assays for naturally activated Ig-secreting cells, and in hybridomas derived by direct fusion of spleen cells from unmanipulated animals. The results show that a very high frequency of splenic resting B cells and plasma cells in transgenic animals produce IgM with B1.8-cross-reactive idiotypes. This was confirmed by hybridoma analysis which also established that the levels of transgene expression and of idiotype-positive IgM production by the same cell are not correlated. The affinities of idiotype-positive endogenous Ig varied, but were generally several orders of magnitude lower than the transgene-encoded idiotype. V regions from idiotype-cross-reactive IgM heavy chains showed marked diversity in sequences that were all different from the transgenic B1.8. These results are compatible with idiotypic mimicry resulting from intercellular selection based on degenerate, whole V region reactivities.

Amino Acid Sequence↗
PubMed1997

Characteristic generated alterations of autoantibody patterns in idiopathic thrombocytopenic purpura.

Using a Western blot technique that allows quantitative detection of antibody reactivities to a large number of antigens, serum IgG and IgM antibody repertoires were compared in a group of 19 patients with a diagnosis of idiopathic thrombocytopenic purpura (ITP) and respective healthy controls. The results show that, irrespective of the duration of thrombocytopenia, age of the patients, and type of therapy, all ITP donors share characteristic alterations of serum antibody reactivity patterns on homologous erythrocyte and liver antigens. Multiparametric analyses of the immunoreactivity data readily segregated the groups of ITP and healthy donors. Similar analyses also distinguished ITP sera from those of a group of patients with systemic lupus erythematosus (SLE). We conclude that ITP is an autoimmune disease associated with generalized alterations of antibody repertoires, that may be characteristic enough to allow for diagnosis.

Adolescent↗
PubMed1997

Neonatal tolerance to alloantigens is induced by enriched antigen-presenting cells.

Spleen and bone marrow cells from normal or Rag-1-deficient donors are equally competent in their ability to induce neonatal transplantation tolerance in semi-allogeneic hosts, and the latter are also capable of tolerizing fully allogeneic recipients. Both types of donor cells resulted in comparable levels of haemopoietic chimerism in tolerant animals. Lymphoid hyperactivity, however, was absent in animals tolerized with Rag-1-deficient cells. The authors conclude that neonatal tolerance induced with haemopoietic cells requires no donor lymphocytes, and is thus not the result of deficient antigen presentation. Furthermore, the state of tolerance can be dissociated from the lymphoid hyperactivity that requires donor lymphocytes and is regularly scored in conventionally tolerized animals.

Animals↗
PubMed1997

Instability of natural antibody repertoires in systemic lupus erythematosus patients, revealed by multiparametric analysis of serum antibody reactivities.

Recent views on autoimmune diseases invoke generalized but specific perturbations in antibody repertoires, rather than the clonally restricted or non-specific polyclonal alterations proposed thus far. The present experiments analyse serum antibody reactivities in 24 systemic lupus erythematosus (SLE) patients and 17 healthy controls, using a method that quantitatively scores a large number of antibody reactivities and allows for multiparametric statistical analyses. The results show global but relatively specific perturbations in SLE antibody repertoires, and identify novel disease-associated reactivity patterns. Furthermore, a time series analysis of serum antibodies over 3 months demonstrates instability of natural antibody repertoires in individual SLE patients, contrasting with their remarkable conservation in healthy donors. Moreover, the method used clusters controls and patients independently, and might prove of diagnostic value, once large data bases are established.

Adolescent↗
PubMed1997

Distinguishable patterns of connectivity in serum immunoglobulins from SLE patients and healthy individuals.

Given that normal individuals maintain significant levels of serum autoantibodies that share many characteristics with those found in association with autoimmune diseases (AID), it has been proposed that disease could result from defects in supraclonal regulation, namely deviations of normal patterns of immunoglobulin (Ig) connectivity. Using conventional methods, together with a recently developed technique to quantitatively score a variety of V-region-dependent serum IgG interactions, the authors have now compared serum Ig connectivity in a group of patients with systemic lupus erythematosus (SLE) to healthy controls. The results demonstrate the existence of V-region interactions of serum IgG and IgM in SLE patients and healthy donors, with comparable connectivity titres, diversity and average affinities (microM range), but a wider individual variation and a tendency for higher F(ab')2 directed reactivities in the group of SLE patients. Multivariate statistics analysis of the data derived from reactivity patterns on F(ab')2 subsets, however, distinguished the two groups of donors, and demonstrated a larger dispersion and wider time-dependent variations in the patient population, as compared to healthy controls. The authors conclude that SLE is associated with circulating antibody repertoires that deviate from the patterns and levels of V-region connectivity characteristic of healthy individuals. These findings may shed light on the mechanisms of disease maintenance, and on the basis for the therapeutic effects of normal polyclonal Igs at high doses.

Adolescent↗
PubMed1997

B lymphocyte sensitivity to IgM receptor ligation is independent of maturation stage and locally determined by macrophage-derived IFN-beta.

Compartmentation of B lymphocyte populations is associated with differences in both development stage and sensitivity to Ig (sIg)-dependent triggering. In order to characterize the factors that contribute in setting the level of sensitivity of a B cell, we quantified sIgM-dependent regulation of Ig secretion in purified mature and immature B cells after ex vivo and in vivo modification of their environment. These analyses formally demonstrate that the bone marrow (BM) microenvironment locally induces high B cell sensitivity to sIgM ligation irrespective of differentiation stage. We further provide evidence that BM macrophages create a dominant environment that enhances B cell sensitivity to B cell receptor triggering. Finally, using ex vivo assays as well as type I IFN receptor-deficient mice we show that IFN-beta produced by resident BM macrophages is necessary and sufficient to define B cell sensitivity. Implications of these findings for the understanding of B cell selection processes are discussed.

Animals↗
PubMed1997

Differential sensitivity of B lymphocyte populations to IgM receptor ligation is determined by local factors.

Ligation of surface IgM on B cells responding to lipopolysaccharide (LPS) suppresses terminal differentiation and high-rate Ig secretion with no effect on proliferation. As shown here, different B cell populations show characteristic mean values of ligand concentration required for 50% inhibition, with Gaussian distributions of sensitivity to IgM receptor ligation that reflect cellular heterogeneity of 'al-or-none' inhibitions in single cells. Differential sensitivity of B cell populations to IgM ligation seems to be locally determined by the cellular environment and unrelated to the 'maturity' of the responding cells. Thus, while long-lived peritoneal B cells are 3- to 5-fold more resistant than splenic B cells, there is no difference in sensitivity between short- and long-lived B cells in the spleen. Furthermore, while B cells in bone marrow and spleen differ in sensitivity by two orders of magnitude, B cells differentiated in vitro from bone marrow pre-B cells are as resistant as splenic B cells. Moreover, bone marrow cell culture supernatants restore a high level of sensitivity in such cell populations. Differential sensitivity to IgM receptor ligation is reproduced by multivalent nominal antigen, in cell populations that show identical dose-response inhibition curves to direct activation of protein kinase C by phorbol esters. We conclude that the level of sensitivity to IgM ligation is independent of the life span or maturity of the B cell, but differentially regulated in vivo by putative tissue factors.

Animals↗
PubMed1996

A model of the immune network with B-T cell co-operation. I--Prototypical structures and dynamics.

Hitherto, "second generation" network models of the immune system have all been restricted to B-lymphocytes and the Ig molecules they produce. These models have not so far been able to provide a convincing mechanism for the distinction between a "Central Immune System" (CIS) composed of a connected network of lymphocyte clones which couple with "self" antigens in a tolerant mode, and a "Peripheral Immune System" (PIS) composed of clones with little or no supra-clonal organization and which produce classical immune responses when interacting with "non-self" antigens. Here, we present a new network model which explicitly incorporates B-T cell co-operation. In this model, B-cell activation is dependent on T-cell help, and activated T-cells are down-regulated by engagement of their TCRs by soluble Ig. We discuss the underlying biology on which we base the system of ordinary differential equations which defines the present network model. We then illustrate some basic features of the model by examining several prototypical situations with a small number of clones. Depending on the idiotypic connectivity structure, the model exhibits two distinct modes of coupling with antigens: an "immune response" mode in which T- and B-cell clones grow exponentially; and a "tolerant" mode in which T-cell clones are controlled by inclusion of all TCRs in the repertoire of an idiotypic B-cell network. Finally, we discuss the simplifying assumptions of the present model and argue that its range of validity is indeed the region of the state-space of the system where the discrimination between the CIS and the PIS take place.

B-Lymphocytes↗