Toll-like receptor 2 activators modulate oral tolerance in mice.

BACKGROUND: Toll-like receptor 2 (TLR2) is a widely expressed pattern recognition receptor critical for innate immunity. TLR2 is also a key regulator of mucosal immunity implicated in the development of allergic disease. TLR2 activators are found in many common foods, but the role of TLR2 in oral tolerance and allergic sensitization to foods is not well understood. OBJECTIVE: The purpose of this study was to evaluate the impacts of TLR2 expression and TLR2 activation on oral tolerance to food antigens in a murine model. METHODS: Mice were fed ovalbumin (OVA) or peanut butter with or without the addition of low doses of TLR2 activators Pam3 CSK4 or FSL-1. Oral tolerance was assessed by analysing antibody responses after a systemic antigen challenge. OVA-specific Tregs were assessed in the Peyer's patches, mesenteric lymph nodes, and spleen in wild-type and TLR2(-/-) mice. Low-dose Pam3 CSK4 was also tested as an oral adjuvant. RESULTS: Oral tolerance was successfully induced in both wild-type and TLR2(-/-) recipient mice, with an associated regulatory T-cell response. Oral TLR2 activation, with low-dose Pam3 CSK4 or FSL-1, during oral antigen exposure was found to alter oral tolerance and was associated with the development of substantial IgE and IgA responses to foods upon systemic challenge. Low-dose oral Pam3 CSK4 treatment also selectively enhanced antigen-specific IgA responses to oral antigen exposure. CONCLUSIONS AND CLINICAL RELEVANCE: TLR2 is not necessary for oral tolerance induction, but oral TLR2 activation modulates humoral IgE and IgA responses during tolerance development. Low-dose Pam3 CSK4 is also an effective oral adjuvant that selectively enhances IgA production. These observations are pertinent to the optimization of oral allergen immunotherapy and oral vaccine development.

Prospective study of infantile haemangiomas: incidence, clinical characteristics and association with placental anomalies.

BACKGROUND: The aetiology and exact incidence of infantile haemangiomas (IHs) are unknown. Prior studies have noted immunohistochemical and biological characteristics shared by IHs and placental tissue. OBJECTIVES: We investigated the possible association between placental anomalies and the development of IHs, as well as the demographic characteristics and other risk factors for IHs. PATIENTS AND METHODS: Pregnant women (n = 578) were prospectively enrolled and their offspring followed for 9 months. Placental evaluations were performed and demographic data collected on all mother-infant pairs. RESULTS: We evaluated 594 infants: 34 haemangiomas [either IH or congenital (CH)] were identified in 29 infants, yielding an incidence of 4·5% for IH (27 infants) and 0·3% for CH (two infants). Placental anomalies were noted in almost 35% of haemangioma-related pregnancies, approximately twice the incidence noted in pregnancies with unaffected infants (P = 0·025). Other risk factors for IH included prematurity (P = 0·016) and low birth weight (P = 0·028). All IHs were present by 3 months of age, and cessation of growth had occurred in all by 9 months of age. Most occurred on the trunk. Of note, 20% of identified IHs were abortive or telangiectatic in nature, small focal lesions that did not proliferate beyond 3 months of age. Only one IH required intervention. CONCLUSIONS: This is the first prospective American study to document the incidence of IHs in infants followed from birth to early infancy. The association with placental anomalies was statistically significant. The overall incidence mirrors prior estimates, but the need for treatment was lower than previously reported.

Effect of particle collisions and aggregation on red blood cell passage through a bifurcation.

Blood flow through bifurcating vessels in the microvasculature leads to uneven distribution of red blood cells (RBC) in the downstream channels when the channels have different sizes or the flow rates through the channels are different. This phenomenon, known as plasma skimming, is responsible for the large variation in hematocrit throughout the circulatory system. Furthermore, the strong streamline curvature present within bifurcations leads to frequent collisions between the blood elements (red and white blood cells and platelets) and the vessel walls, as well as a rearrangement of the distribution of the blood elements in the channels downstream of the bifurcation. Computational models of bifurcation flows typically neglect collision and adhesion of RBCs with each other. In this paper, we use a new type of discrete-element model to investigate the effect of particle-particle collisions and RBC aggregation on modeling of plasma skimming in bifurcations. Cases are examined with and without RBC adhesion and for different hematocrit values, including validation against previous computational results. Results show significant plasma skimming in the low-flow daughter branch and an increase in fractional particle flux with increased hematocrit, as in experimental studies. Accounting for particle-particle collisions leads to a considerable increase in collision rate of particles with the vessel wall. Particles are found to approximately follow fluid velocity streamlines, and consequently particle-particle collisions and aggregation do not significantly affect plasma skimming.

Thyroxine-binding globulin: characterization of the binding site with a fluorescent dye as a probe.

The fluorescent dye 1,8-anilinonaphthalenesulfonate competed with thyroxine for binding to thyroxine-binding globulin. Fluorescence analysis indicated that the dye bound to the globulin in a molar ratio of 1:1 and with an association constant (at 23 degrees C) of 4.19 x10(6)M(-1), and that thyroxine bound to the globulin in a molar ratio of 1:1 and with an association constant (at 23 degrees C) of 2.35x10(10)M(-1). Displacement of globulin-bound dye by thyroxine was shown by fluorescence quenching, and displacement of globulin-bound thyroxine by dye was demonstrated by ultrafiltration.

Studies on human thyroxine-binding globulin (TBG). I. Purification of TBG and immunologic studies on the relationship between TBG from normal persons and those with TBG "deficiency".

A method for obtaining highly purified thyroxine-binding globulin (TBG) from whole human serum is presented. The method employs relatively simple procedures of step-wise ammonium sulfate precipitation followed by column chromatography on DEAE cellulose and DEAE Sephadex. The final product produces a single protein band on disc electrophoresis. The sedimentation constant of the TBG thus purified is 3.91 and its calculated mol wt is 54,000. An antiserum to the highly purified TBG produced a single arc on immunoelectrophoresis. When the antiserum was reacted against normal human serum or against serum from subjects deficient in TBG, each produced two arcs-one identical with that produced by the antigen alone. The second arc is probably the result of a contaminating protein in the antigen, present in too low a concentration to be detectable by disc gel electrophoresis. It is concluded that some persons with TBG "deficiency" have a circulating protein, immunologically indistinguishable from TBG, which is defective in its ability to bind thyroxine.

Studies on human thyroxine-binding globulin (TBG). IX. Some physical, chemical, and biological properties of radioiodinated TBG and partially desialylated TBG.

Thyroxine-binding globulin (TBG) and partially desialylated or slow TBG (STBG) were purified from human serum by affinity chromatography. Purified TBG was identical to TBG present in serum by the criteria of electrophoretic mobility, affinity for thyroxine (T4), and heat-inactivation response. Purified STBG had slower electrophoretic mobility and lower affinity for T4. Both bound T4 in an equimolar ratio, were immunoprecipitable, and had similar inactivation t1/2 at 61 degrees C. TBG and STBG were iodinated by the chloramine-T-catalyzed reaction. An average of from 0.02 to 6 atoms I could be incorporated per molecule of the protein by adjusting the conditions of the reaction (time, protein and iodide concentrations). 125-I, 131-I, and 127-I were used. Iodination increased the anodal mobility of TBG but did not affect the reversible T4-binding, precipitation by antiserum, or the heat-inactivation properties. "Heavily" and "lightly" iodinated TBG had identical disappearance half-times from serum in the rabbit. 15 min after the intravenous administration of [131-I]-STBG and [125-I]TBG mixture to rats, more than 90% of the injected 131-I dose was in the liver, and the liver 131-I/125-I ratio was 32-fold that of serum. Selective uptake of STBG by the liver was also observed in the rabbit and in man. The serum [125-I]STBG/[131-I]TBG ratio declined from 1 to 0.2 in 10 min in the intact rabbit but remained unchanged for 1 h in the acutely hepatectomized animal. In the rabbit, t 1/2 was approximately 3 min for STBG and 0.8-3.4 days for TBG. The radioiodine derived from the iodinated proteins is partly excreted in bile but the bulk was precipitable with specific antibodies. Some isotope in the form of iodide appeared in blood and was excreted in the urine. Since radioiodinated TBG and STBG preserve their biologic and immunologic properties they are useful as tracer materials for metabolic studies. In rat, rabbit, and man STBG is rapidly cleared from serum by the liver. Conversion of TBG to STBG may be the limiting step in the regulation of TBG metabolism.

Studies on human thyroxine-binding globulin. VI. The nature of slow thyroxine-binding globulin.

A model system utilizing a highly purified partially desialylated thyroxine-(T(4)) binding protein (STBG) was studied. STBG was prepared by the same affinity chromatographic method we have reported for preparation of highly purified T(4)-binding globulin (TBG). The necessary prerequisite for preparation of STBG was the use of T(4)-substituted Sepharose which had been repeatedly exposed to large volumes of serum for purification of TBG. STBG moved more slowly on cellulose acetate electrophoresis than TBG but had the same molecular weight and antigenic determinants as TBG. It bound T(4) with a 1: 1 molar ratio but its affinity for T(4) was about 10 times less than that of TB. STBG had about onefourth the sialic acid content of TBG and the electrophoretic mobility of this protein was similar to that of a T(4)-binding protein with a mobility slower than that of TBG which has been seen in the electrophoretic patterns of some normal human serums and in serums of patients with hepatic cirrhosis and which does not appear to be an artifact caused by storage and freezing of serum. This fourth slowly migrating T(4)-binding region in electrophoretograms of cirrhotic serums is completely abolished by prior incubation with rabbit antiserum to TBG. The in vitro production of partially desialylated TBG from T(4)-Sepharose which had been previously exposed to large volumes of serum may be due to adsorption of neuraminidases to the Sepharose either directly from serum or as the result of bacterial contamination. Partial desialylation of TBG in vivo may be an early step in the catabolism of this protein.

Interleukin (IL)-10 inhibits long-term IL-6 production but not preformed mediator release from rat peritoneal mast cells.

Mast cells have been implicated in a number of diseases involving chronic inflammation including asthma, rheumatoid arthritis, and inflammatory bowel diseases. They are a potent source of several cytokines, including IL-6 and TNF-alpha. Freshly isolated rat peritoneal mast cells will produce IL-6 in response to anti-IgE, LPS, PGE1, or PGE2; however, the mechanisms by which such cytokine production is regulated are poorly understood. IL-10 is recognized as an important immunoregulatory cytokine with effects on T cell development and the production of inflammatory cytokines. IL-10 has previously been described to enhance mast cell development in the context of IL-3 and IL-4. In the current study, we have examined the ability of IL-10 to modulate rat peritoneal mast cell IL-6 and TNF-alpha production in response to a variety of stimuli. We have observed that recombinant murine IL-10 can inhibit the production of both IL-6 and TNF-alpha by mast cells without altering the degree of histamine release in response to anti-IgE. Concentrations of IL-10 as low as 0.2 ng/ml were sufficient to inhibit IL-6 production by LPS- or anti-IgE-activated cells significantly. IL-10 also inhibited PGE1- and PGE2-induced IL-6 production. The relative potency of IL-10 as an inhibitor of mast cell IL-6 production was highly dependent upon the stimulus used, with a 10-fold difference in the IC50 for LPS- or anti-IgE-activated cells (0.21 ng/ml) and cells activated with a combination of LPS and PGE2 (2.29 ng/ml). This suggests that prostanoids may limit the ability of IL-10 to modulate mast cell IL-6 production in the context of inflammation. These data have important implications for the regulation of mast cell IL-6 in inflammatory diseases involving prostanoid production and the effects of treatment with cyclooxygenase inhibitors. Our results also demonstrate a dual role for IL-10 on mast cells as a growth factor and inhibitor of cytokine production.

Partial purification of plasma thromboplastin antecedent (factor XI) and its activation by trypsin.

A persistent puzzle in our understanding of hemostasis has been the absence of hemorrhagic symptoms in the majority of patients with Hageman trait, the hereditary deficiency of Hageman factor (factor XII). One proposed hypothesis is that alternative mechanisms exist in blood through which plasma thromboplastin antecedent (PTA, factor XI) can become active in the absence of Hageman factor. In order to test this hypothesis, the effect of several proteolytic enzymes, among them thrombin, plasma kallikrein, and trypsin, was tested upon unactivated PTA. PTA was prepared from normal human plasma by Ca(3)(PO(4))(2) adsorption, ammonium sulfate fractionation, and successive chromatography on QAE-Sephadex (twice). Sephadex-G150, and SP-Sephadex. The partially purified PTA was almost all in its native form, with a specific activity of 45-70 U/mg protein; the yield was about 10%. It contained no measurable amounts of other known clotting factors, plasmin, plasminogen, nor IgG. Incubation of PTA with trypsin generated potent clot-promoting activity that corrected the abnormally long clotting time of plasma deficient in Hageman factor or PTA but not in Christmas factor. This clot-promoting agent behaved like activated PTA on gel filtration (apparent molecular weight: 185,000) and was specifically inhibited by an antiserum directed against activated PTA. These data suggested that PTA can be converted into its active form by trypsin. PTA was not activated by thrombin, chymotrypsin, papain, ficin, plasmin, plasma kallikrein, tissue thromboplastin, or C. Trypsin converted PTA to its active form enzymatically. Whether trypsin serves to activate PTA in vivo is not yet clear.

Metabolism of thyroxine-binding globulin in man. Abnormal rate of synthesis in inherited thyroxine-binding globulin deficiency and excess.

It has been previously suggested that inherited thyroxine-binding globulin (TBG) abnormalities in man may be due to mutations at a single X-chromosome-linked locus controlling TBG synthesis. However, abnormalities in TBG degradation have not been excluded. The availability of purified human TBG and its successful labeling with radioiodide allowed us to examine such possibility. Human TBG was purified by affinity chromatography, labeled under sterile conditions with 131I or 125I,, and mixed with [125I]thyroxine (T4) or [131I]T4, respectively, before their intravenous injection. Blood and urine samples were collected over a 10-day period, and the turnover parameters were calculated. In eight normal volunteers mean values +/-SD for TBG and T4 respectively, were as follows: Half time (t1/2) 5.3 +/- 0.4 and 7.0 +/- 0.6 days; distribution space (DS) 7.2 +/- 1.0 and 10.8 +/- 1.2 liters; and total daily degradation (D) 0.211 +/- 0.053 and 0.088 +/- 0.011 mumol/day. In all subjects, t1/2 of TBG was shorter than that of T4; and the DS was smaller. 2.4 mol of TBG was degraded for each mole of T4. In five of six subjects from four families, comprising hemizygous and heterozygous carriers of TBG absence, decrease, and excess, the t1/2 and DS for TBG were within the normal range. The D of TBG was proportional to the serum concentration of the protein. Changes in the T4 kinetics in these patients were compatible with euthyroidism and with the known alterations in the extrathyroidal T4 pool associated with the changes in serum TBG concentration. A striking decrease in the t1/2 of TBG was found only in a patient with acquired diminution in TBG concentration and in patients with thyrotoxicosis or other conditions apparently unrelated to thyroid dysfunction. TBG t1/2 was 2.5 days in a patient with multiple myeloma and 3.6 days in two patients with thyrotoxicosis. Decreased TBG t1/2 was also observed in three of six patients with nonthyroidal pathology and was associated with an increase in TBG D disproportionate to their level of serum TBG. These studies indicate that changes in TBG concentration in patients with X-chromosome-linked TBG abnormalities are due to alterations in its rate of synthesis. In other conditions, abnormalities of TBG degradation and/or rate of synthesis may be found.

Measurement of circulating desialylated glycoproteins and correlation with hepatocellular damage.

Addition of increasing amounts of (125)I-labeled desialylated thyroxine-binding globulin (DTBG) to hepatic cell membranes resulted in a progressive increase in binding. Saturability of membrane sites was indicated by a concentration beyond which further increases in [(125)I]DTBG resulted in no further binding. The binding curve for [(125)I]DTBG was similar to binding curves of desialylated orosomucoid, fetuin, and ceruloplasmin. An inhibition assay system using hepatic cell membranes showed that desialylated orosomucoid had a greater affinity for membrane binding sites than did DTBG but desialylated fetuin and ceruloplasmin bound less avidly than DTBG. Serum from normal persons and patients with a variety of illnesses was tested for its ability to inhibit [(125)I]DTBG binding. The inhibitory activity of 1 ml of normal serum was equivalent to that of 0.2-2 mug DTBG. Patients with Laënnec's cirrhosis, biliary cirrhosis, and hepatic metastases had greatly increased inhibitory activity in their serum. Patients with jaundice due to extrahepatic obstruction had inhibitory activity not significantly different from that found in normal serum. Column chromatography of normal serum on Sephadex G-200 resulted in inhibitory activity throughout the range of protein molecular weight. Desialylation of normal serum with neuraminidase enhanced the inhibitory activity but did not change the distribution of the activity. Gel chromatography of cirrhotic serum showed markedly increased inhibitory activity associated with the macroglobulins and the 4.5S peak and a new peak of inhibitory activity in the low molecular weight area was also seen. Inhibition of desialylated glycoprotein binding to liver cell membranes by serum from patients with hepatocellular disease raises the possibility that desialylated serum glycoproteins accumulate in the circulation and that patients with compromised hepatocellular function may no longer be able to clear them from the circulation. Alternatively, accumulation of desialylated glycoproteins in the circulation could result from defective protein synthesis by the diseased liver.

The role of mast cells in inflammatory reactions of the airways, skin and intestine.

The concept that mast cells play a key role in the initiation of acute allergic responses has been around for many years. However, the role of mast cells in the chronic processes that are the hallmark of inflammatory disease is still poorly understood. With better techniques to study mast cell function it has become clear that these cells may have a much wider role in immune responses and regulation than previously recognized. Exciting progress has been made over the past year in defining the breadth of mast cell functions in inflammation. Further studies are necessary to evaluate the in vivo significance of many of these findings.

Expression of the C4 Me1 Gene from Flaveria bidentis Requires an Interaction between 5[prime] and 3[prime] Sequences.

The efficient functioning of C4 photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle sheath cells. To determine the mechanism controlling bundle sheath cell-specific expression of the NADP-malic enzyme, we made a set of chimeric constructs using the 5[prime] and 3[prime] regions of the Flaveria bidentis Me1 gene fused to the [beta]-glucuronidase gusA reporter gene. The pattern of GUS activity in stably transformed F. bidentis plants was analyzed by histochemical and cell separation techniques. We conclude that the 5[prime] region of Me1 determines bundle sheath specificity, whereas the 3[prime] region contains an apparent enhancer-like element that confers high-level expression in leaves. The interaction of 5[prime] and 3[prime] sequences was dependent on factors that are present in the C4 plant but not found in tobacco.

Role of pituitary hormones in the growth of human breast cancer.

Hypophysectomy was performed in 28 women with Stage IV breast cancer who were treated initially with antiestrogens. Six of 13 patients who responded to tamoxifen and 2 of 12 who failed to benefit from tamoxifen obtained remissions from hypophysectomy. The remissions average 11+ months. Three of 8 patients treated initially with antiestrogens have responded to androgen therapy. The results suggest that hormones other than estrogen, which appears to play a major role, may be involved in stimulating the growth of some human breast cancers. Prolactin receptors were detectable in 51% of human breast cancers and were detected in both estrogen receptor-positive and -negative tumors. Preliminary clinical correlations suggest that prolactin receptors will not be useful in predicting response to antiestrogen therapy.

Serum inhibitors of desialylated glycoprotein binding to hepatocyte membranes.

Identification of the material present in human serum which is responsible for inhibition of binding of desialylated glycoproteins to rat hepatocyte membranes was accomplished by means of affinity chromatography using Sephadex to which the galactose-specific lectin, Ricinus Communis Agglutinin (RCAI) was covalently bound. RCAI-Sephadex was capable of extraction of virtually all of the inhibitory activity from cirrhotic serum. The RCA I-bound inhibitory activity could be eluted with 0.05 M D-galactose. The D-galactose eluate when subjected to radioimmunoelectrophoresis against a number of specific antibodies to human serum glycoproteins produced arcs corresponding to alpha 1-acid glycoprotein, alpha2-macroglobulin, IgG, IgA, and IgM. In another experiment putative terminal galactosyl groups of desialylated glycoproteins in the D-galactose eluate from cirrhotic serum exposed to RCAI-Sephadex were labelled with tritiated borohydride after treatment with galactose oxidase. Subsequent gel electrophoresis showed peaks of radioactivity throughout the area of the gel corresponding to protein molecular weights of the 19 S, 7 S, and 4 S classes. It thus appears that a heterogeneous population of desialylated serum glycoproteins accounts for the inhibition of binding of desialylated glycoprotein to the hepatocyte membrane and that these desialylated glycoproteins are present in small amounts in normal human serum and in greatly increased quantities in serum from patients with cirrhosis.

Cell surface changes in diabetic rats. Studies of lectin binding to liver cell plasma membranes.

We have previously reported changes in the chemical composition of cell surface membranes in diabetic rats (Chandramoulis, V. and Carter, Jr., J. R. (1975) Diabetes 24, 257-262 [1]). To examine the possible implications of these changes for cell surface structures, we have measured the binding of labeled lectins and desialylated glycoproteins to plasma membranes prepared from the livers of streptozotocin--diabetic and control rats. Lectins were chosen which have affinities for different carbohydrate moieties. The binding of ricin and concanavalin A to liver cell membranes from the diabetic rats was significantly reduced, but no change in the binding of wheat germ agglutinin was noted. Binding of desialylated thyrozine--binding globulin, previously shown to be dependent on membrane sialic acid residues, ws strongly suggest that insulin deficiency leads to generalized changes in cell surfaced glycoproteins, at least in this animal model of diabetes.

CpG-containing oligodeoxynucleotides induce TNF-alpha and IL-6 production but not degranulation from murine bone marrow-derived mast cells.

Mast cells are sentinel cells critical to the initiation of innate immune and inflammatory responses, particularly at mucosal surfaces. To fulfill this function they can be activated by several pathogen-associated stimuli to produce cytokines with or without concurrent degranulation. We examined the ability of immunostimulatory DNA sequences including CpG motifs, which are found in increased quantities in bacterial DNA, to activate mouse bone marrow-derived mast cells (mBMMC). Mast cells were treated with a range of doses of CpG-containing oligodeoxynucleotides or control oligodeoxynucleotides without CpG within their sequence. There was a dose-dependent increase in the production of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) by mast cells treated with the CpG-containing oligodeoxynucleotides. The cytokine levels induced were directly related to the number of CpG within a given length of sequence. Treatment with oligonucleotides containing 3CpG induced an eightfold increase in TNF production over control incubated mast cells. Other cytokines, including granulocyte-macrophage colony-stimulating factor, IL-4, interferon-gamma, and IL-12 were not induced by oligonucleotide treatment. Neither CpG containing oligodeoxynucleotides nor control oligodeoxynucleotides induced degranulation of mast cells. Bacterial DNA from Escherichia coli also induced IL-6 from mBMMC but neither calf thymus DNA nor methylase-treated E. coli DNA had such an effect. Examination of the uptake of Texas red-labeled CpG and non-CpG-containing oligodeoxynucleotides revealed that they were both similarly taken up by the mBMMC. These results have important implications for the mechanism by which mast cells respond to bacteria and for the potential role of mast cells in DNA vaccination.

Toll-like receptor 4-mediated activation of murine mast cells.

Toll-like receptors (TLRs) are a family of pattern recognition receptors that are critical for cellular responses to a variety of bacterial, viral, and fungal products. Mast cells are important to host survival in a number of models of bacterial infection and might act as sentinel cells in host defense. We therefore examined the expression of TLRs and associated molecules by murine bone marrow-derived mast cells (BMMCs). BMMCs and the murine mast cell line MC/9 expressed mRNA for TLR2, TLR4, and TLR6 but not TLR5 and for both adapter molecule MD-2 and signaling molecule MyD88 but lacked surface CD14. After activation with the TLR2- and TLR4-dependent stimuli Staphylococcus aureus-derived peptidoglycan and Escherichia coli-derived lipopolysaccharide (LPS), respectively, mast cells produced significant levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). To determine whether mast cells require TLR4 for cellular responses to LPS, mast cells were derived from the bone marrow cells of C3H/HeJ and C57Bl/10ScNCr mice containing a point mutation and a null mutation, respectively, in TLR4. Using these models, we demonstrated that the BMMC IL-6 and TNF-alpha responses to LPS were completely dependent on functional TLR4 with no significant LPS response observed in its absence. These findings have important implications for the mechanism of mast cell responses to pathogens and their products and suggest that different TLR4-expressing cells might have different thresholds for activation with LPS.

Selective antibody blockade of lymphocyte migration to mucosal sites and mast cell adhesion.

The integrins alpha4beta7 and alpha4beta1 mediate adhesion to the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and the vascular cell adhesion molecule-1 (VCAM-1) and are important in T cell and allergic inflammatory reactions in the rat. The relative contributions of alpha4beta7 and alpha4beta1 in these reactions is unknown. To examine the role of alpha4beta7 in the rat a new mAb, TA-6, was developed. TA-6 inhibited adhesion to MAdCAM-1 but not to VCAM-1, a characteristic of alpha4beta7 adhesion, and immunofluorescence and immunoprecipitation studies were compatible with binding to alpha4beta7. TA-6 blocked rat lymphocyte adhesion to mesenteric lymph nodes and T cell migration to mucosal lymphoid tissues and it bound to rat mucosal mast cells. TA-6 did not inhibit lymphocyte adhesion to peripheral lymph nodes and T cell migration to peripheral lymphoid tissues or cutaneous inflammatory sites, and was not expressed on connective tissue mast cells.

CD43-, but not CD43+, IL-10-producing CD1dhiCD5+ B cells suppress type 1 immune responses during Chlamydia muridarum genital tract infection.

Regulatory B (Breg) cells are known to modulate immune responses through predominantly interleukin-10 (IL-10)-dependent mechanisms and can be hypothetically divided into innate and adaptive subsets based on the nature of their activating signals. However, the specific role of different Breg subsets in modulating immune responses remains ambiguous. Here we have shown that Chlamydia induces IL-10-producing splenic B-cell populations consisting of CD43(+) and CD43(-) subsets of IgM(hi)IgD(lo) innate-like B (ILB) cells in vitro. While CD43(+)IL-10-producing B cells displayed innate type features and were readily induced by Chlamydia via Toll-like-receptor (TLR) signaling, CD43(-)IL-10-producing B cells required additional B-cell activating factor (BAFF)-mediated signals from dendritic cells (DCs) for their differentiation and activation, thereby classifying them as adaptive type Bregs. Importantly, CD43(-), but not CD43(+), IL-10-producing ILB cells displayed bona fide Breg activity by potently suppressing interferon-γ (IFN-γ) production in vitro in an IL-10-dependent manner. Furthermore, a novel CD43(-)CD1d(hi)CD5(+) IL-10-producing Breg population was predominantly induced by Chlamydia genital infection in vivo. Correspondingly, mixed bone marrow chimeric mice with B-cell-specific IL-10 deficiency exhibited significantly increased type 1 immune responses, decreased bacterial burden, and reduced oviduct pathology upon infection. Our data demonstrate for the first time a distinct role for CD43(-)CD1d(hi)CD5(+)-adaptive Bregs over CD43(+) innate counterparts in controlling mucosal responses against intracellular bacterial infection.