Functional effects of TGF-ß1 on mesenchymal stem cell mobilization in cockroach allergen-induced asthma.

Mesenchymal stem cells (MSCs) have been suggested to participate in immune regulation and airway repair/remodeling. TGF-ß1 is critical in the recruitment of stem/progenitor cells for tissue repair, remodeling, and cell differentiation. In this study, we sought to investigate the role of TGF-ß1 in MSC migration in allergic asthma. We examined nestin expression (a marker for MSCs) and TGF-ß1 signaling activation in airways in cockroach allergen extract (CRE)-induced mouse models. Compared with control mice, there were increased nestin(+) cells in airways and higher levels of active TGF-ß1 in serum and p-Smad2/3 expression in lungs of CRE-treated mice. Increased activation of TGF-ß1 signaling was also found in CRE-treated MSCs. We then assessed MSC migration induced by conditioned medium from CRE-challenged human epithelium in air/liquid interface culture in Transwell assays. MSC migration was stimulated by epithelial-conditioned medium, but was significantly inhibited by either TGF-ß1-neutralizing Ab or TßR1 inhibitor. Intriguingly, increased migration of MSCs from blood and bone marrow to the airway was also observed after systemic injection of GFP(+) MSCs and from bone marrow of Nes-GFP mice following CRE challenge. Furthermore, TGF-ß1-neutralizing Ab inhibited the CRE-induced MSC recruitment, but promoted airway inflammation. Finally, we investigated the role of MSCs in modulating CRE-induced T cell response and found that MSCs significantly inhibited CRE-induced inflammatory cytokine secretion (IL-4, IL-13, IL-17, and IFN-γ) by CD4(+) T cells. These results suggest that TGF-ß1 may be a key promigratory factor in recruiting MSCs to the airways in mouse models of asthma.

Aryl hydrocarbon receptor controls murine mast cell homeostasis.

We propose that the aryl hydrocarbon receptor (AhR), a unique chemical sensor, is critical in controlling mast cell differentiation, growth, and function in vitro and in vivo. In antigen-stimulated mast cells, exposure to AhR ligands resulted in a calcium- and reactive oxygen species (ROS)-dependent increase of reversible oxidation in and reduced activity of SHP-2 phosphatase, leading to enhanced mast cell signaling, degranulation, and mediator and cytokine release, as well as the in vivo anaphylactic response. Surprisingly, significant mast cell deficiency was noted in AhR-null mice due to defective calcium signaling and mitochondrial function, concomitant with reduced expression of c-kit and cytosolic STAT proteins, as well as enhanced intracellular ROS and apoptosis. Consequently, AhR-null mast cells responded poorly to stimulation, demonstrating a critical role of AhR signaling in maintaining mast cell homeostasis.

Aryl hydrocarbon receptor (AhR) modulates cockroach allergen-induced immune responses through active TGFß1 release.

BACKGROUND: Aryl hydrocarbon receptor (AhR), a multifunctional regulator that senses and responds to environmental stimuli, plays a role in normal cell development and immune regulation. Recent evidence supports a significant link between environmental exposure and AhR in the development of allergic diseases. We sought to investigate whether AhR plays a role in mediating cockroach allergen-induced allergic immune responses. METHODS: AhR expression in human lung fibroblasts from asthmatic and healthy individuals and in cockroach extract (CRE) treated human lung fibroblasts (WI-38) was examined. The role of AhR in modulating CRE induced TGFß1 production was investigated by using AhR agonist, TCDD, antagonist CH122319, and knockdown of AhR. The role of latent TGFß1 binding protein-1 (LTBP1) in mediating TCDD induced active TGFß1 release was also examined. RESULTS: AhR expression was higher in airway fibroblasts from asthmatic subjects as compared to healthy controls. AhR in fibroblasts was activated by TCDD with an increased expression of cyp1a1 and cyp1b1. Increased AhR expression was observed in CRE-treated fibroblasts. Importantly, CRE induced TGFß1 production in fibroblasts was significantly enhanced by TCDD but inhibited by CH122319. Reduced TGFß1 production was further confirmed in fibroblasts with AhR knockdown. Moreover, AhR knockdown inhibited CRE induced fibroblast differentiation. Furthermore, TCDD induced active TGFß1 release was significantly inhibited by LTBP1 knockdown. CONCLUSION: These results provide evidence for the role of AhR in modulating cockroach allergen-induced immune responses through controlling the active TGFß1 release, suggesting a possible synergistic effect between exposure to allergens and environmental chemicals on the development of allergic diseases.

Functional interaction of common allergens and a C-type lectin receptor, dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN), on human dendritic cells.

Fucosylated glycans on pathogens are known to shape the immune response through their interaction with pattern recognition receptors, such as C-type lectin receptors (CLRs), on dendritic cells (DCs). Similar fucosylated structures are also commonly found in a variety of allergens, but their functional significance remains unclear. To test a hypothesis that allergen-associated glycans serve as the molecular patterns in functional interaction with CLRs, an enzyme-linked immunosorbent assay-based binding assay was performed to determine the binding activity of purified allergens and allergen extracts. THP-1 cells and monocyte-derived DCs (MDDCs) were investigated as a model for testing the functional effects of allergen-CLR interaction using enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. Significant and saturable bindings of allergens and allergen extracts with variable binding activities to DC-specific ICAM3-grabbing non-integrin (DC-SIGN) and its related receptor, L-SIGN, were found. These include bovine serum albumin coupled with a common glycoform (fucosylated glycan lacking the alpha1,3-linked mannose) of allergens and a panel of purified allergens, including BG60 (Cyn dBG-60; Bermuda grass pollen) and Der p2 (house dust mite). The binding activity was calcium-dependent and inhibitable by fucose and Lewis-x trisaccharides (Le(x)). In THP-1 cells and human MDDCs, BG60-DC-SIGN interaction led to the activation of Raf-1 and ERK kinases and the induction of tumor necrosis factor-alpha expression. This effect could be blocked, in part, by Raf-1 inhibitor or anti-DC-SIGN antibodies and was significantly reduced in cells with DC-SIGN knockdown. These results suggest that allergens are able to interact with DC-SIGN and induce tumor necrosis factor-alpha expression in MDDCs via, in part, Raf-1 signaling pathways.

Regulatory role of DC-derived osteopontin in systemic allergen sensitization.

Osteopontin (OPN) is a secreted phosphoglycoprotein with a wide range of functions, and is involved in various pathophysiological conditions. However, the role of OPN in IgE and Th2-associated allergic responses remains incompletely defined. The aim of this study was to elucidate the role of OPN in systemic allergen sensitization in mice. When compared with OPN(+/+) mice, significantly increased levels of OVA-induced IgE were found in OPN(-/-) mice. OPN(-/-) DC demonstrated an increased capacity to enhance Th2 cytokine production in CD4+ T cells from sensitized OPN(+/+) mice. Furthermore, significantly reduced levels of IL-12p70 expression were seen in LPS-stimulated OPN(-/-) DC as compared with the WT DC, and the reduction was reversible by the addition of recombinant OPN (rOPN). rOPN was able to suppress OVA-induced IL-13 production in the cultures of CD4 and OPN(-/-) DC, but this inhibitory activity was neutralized by the addition of anti-IL-12 Ab. In addition, administration of rOPN in vivo suppressed OVA-specific IgE production; however, this suppressive effect was abrogated in IL-12-deficient mice. These results indicate that DC-derived OPN plays a regulatory role in the development of systemic allergen sensitization, which is mediated, at least in part, through the production of endogenous IL-12.

Interleukin-10 and Th2 cytokines differentially regulate osteopontin expression in human monocytes and dendritic cells.

Osteopontin (OPN) is a pleiotrophic phosphoprotein involved in homeostatic and pathophysiologic responses. It is known to be a chemotactic cytokine for dendritic cells (DCs), a critical cell type in both innate and adaptive immune responses. We report herein a contrasting role of interleukin-10 (IL-10) and Th2 cytokines in the regulation of OPN expression in human monocytes and monocyte-derived DCs (Mo-DCs). Our results showed first that the expression of OPN in monocytes and Mo-DCs was induced in a time-dependent and dose-dependent manner by IL-10 but was inhibited by IL-4 or IL-13. Further, the basal level of OPN expression was also inhibited by IL-4. This inhibitory effect of IL-4 was associated with a faster decay of OPN transcripts and a decreased proximal promoter activity of OPN in IL-4-treated cells. These results demonstrate a novel role of IL-10 and Th2 cytokines in the regulation of DC function through their contrasting regulatory activities on the expression of OPN.

Endotoxin contamination contributes to the in vitro cytokine-inducing activity of osteopontin preparations.

Recently, both native and recombinant preparations of human osteopontin (OPN) have been shown to be able to induce the production of several proinflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) or purified monocytes. In the present study, we found that commercially available native and recombinant OPNs contain variable amounts of endotoxin (LPS) and that removal of endotoxin by polymyxin B-agarose column abrogated their cytokine-inducing activity. These results suggest the questionable evidence of the ability of OPN to induce several cytokines in human PBMCs and draw attention to the exquisite sensitivity of PBMCs/monocytes to endotoxin contaminants.

Murine mast cells secrete and respond to interleukin-33.

Interleukin-33 (IL-33) appears to play a crucial role in the expression of allergic diseases, but its cellular source and regulatory mechanisms remain to be fully elucidated. Mast cells, one of the major effecter cell populations in mediating allergy, express high levels of IL-33 receptor, ST2, and have been shown to express IL-33 transcripts. In this study, we aimed to examine the secretion of IL-33 in mast cells and their response to IL-33. We have successfully detected secreted IL-33 from cell supernatants through a modified enzyme-linked immunosorbent assay (ELISA) technique-cell-based ELISA. Activation of bone marrow-derived cultured mast cells (BMMCs) by crosslinkage of an antigen [ovalbumin (OVA)] and OVA-specific IgE mAbs significantly induced the expression of IL-33 transcripts, cytosolic and secreted proteins. In addition, the Toll-like receptor (TLR) 2 and TLR-9 ligands could trigger IL-33 mRNA expression. Exposure of BMMCs to IL-33 significantly increased the levels of IL-13 and IL-6 expression, concomitant with enhanced activation of mitogen-activated protein kinase (MAPKs) (ERK, p38, and JNK) and nuclear factor-kappa B. These results suggest that mouse BMMCs are capable of producing and serving as endogenous sources of IL-33, and that IL-33 plays an important role in regulating mast cell functions.

Functional interaction of cockroach allergens and mannose receptor (CD206) in human circulating fibrocytes.

BACKGROUND: The innate pattern recognition C-type-lectin receptors (CLRs), including mannose receptor (MRC1; CD206), have been suggested to functionally interact with allergens and are critical in controlling immune response. Fibrocytes have been considered to play a role in allergic asthma. Here we sought to investigate the functional interaction of cockroach allergens with CD206 in fibrocytes. METHODS: Profiling of N-linked glycans from natural purified cockroach allergen Bla g 2 was accomplished by MALDI-MS. The binding activity of cockroach allergens to CD206 was determined by solid-phase binding assays. Levels of CD206 expression on human fibrocytes and CD206 mediated signaling and cytokine production in Bla g 2 treated fibrocytes were determined. RESULTS: Profiling of N-linked glycans from Bla g 2 revealed a predominance of small, mannose-terminated glycans with and without fucose. Significant binding of Bla g 2 to CD206 was observed, which was inhibited by yeast mannan (a known CD206 ligand), free mannose, and a blocking antibody (anti-hMR). Flow cytometric analyses of human fibrocytes (CD45(+) and collagen-1(+)) showed selective expression of CD206 on fibrocytes. Functionally, a concentration-dependent uptake of FITC labeled Bla g 2 by fibrocytes was observed, but was significantly inhibited by anti-hMR. Bla g 2 can stimulate up-regulation of inflammatory cytokines including TNF-alpha and IL-6 and activation of nuclear factor kappa B (NF-kB/p65), p38 mitogen-activated protein kinase (p38), ERK, and JNK in cultured fibrocytes. This increased secretion of TNF-alpha and IL-6 and activation of NF-kB, ERK, and JNK was significantly inhibited by the addition of either mannan or mannose. Furthermore, Bla g 2 induced increase in TNF-alpha and IL-6 production was also inhibited by the use of NF-kB, ERK, and JNK inhibitors. CONCLUSION: These results provide evidence supporting the existence of a functional cockroach allergen-CD206 axis in human fibrocytes, suggesting a role for CD206 in regulating allergen induced allergic responses in asthma.

Oral tolerance to food-induced systemic anaphylaxis mediated by the C-type lectin SIGNR1.

We propose that a C-type lectin receptor, SIGNR-1 (also called Cd209b), helps to condition dendritic cells (DCs) in the gastrointestinal lamina propria (LPDCs) for the induction of oral tolerance in a model of food-induced anaphylaxis. Oral delivery of BSA bearing 51 molecules of mannoside (Man(51)-BSA) substantially reduced the BSA-induced anaphylactic response. Man(51)-BSA selectively targeted LPDCs that expressed SIGNR1 and induced the expression of interleukin-10 (IL-10), but not IL-6 or IL-12 p70. We found the same effects in IL-10-GFP knock-in (tiger) mice treated with Man(51)-BSA. The Man(51)-BSA-SIGNR1 axis in LPDCs, both in vitro and in vivo, promoted the generation of CD4(+) type 1 regulatory T (Tr1)-like cells that expressed IL-10 and interferon-γ (IFN-γ), in a SIGNR-1- and IL-10-dependent manner, but not of CD4(+)CD25(+)Foxp3(+) regulatory T cells. The Tr1-like cells could transfer tolerance. These results suggest that sugar-modified antigens might be used to induce oral tolerance by targeting SIGNR1 and LPDCs.

Antigen coupled with Lewis-x trisaccharides elicits potent immune responses in mice.

BACKGROUND: Glycoproteins containing Lewis-x (Le(x)) trisaccharides are often associated with the host's adaptive T(H)2-type immunity, but the mechanisms underlying the T(H)2-biased response are at present unclear. OBJECTIVE: The modulatory effect of Le(x) or its glycoconjugates on IgE/T(H)2 responses was investigated. METHODS: The levels of serum antibodies and cytokines were analyzed by means of ELISA, RT-PCR, or both. RESULTS: In C3H mice Le(x) coupled with BSA (Le(x)-BSA) elicited higher levels of specific IgE and IgG1, but not IgG2a, which were associated with increased levels of splenic T(H)2 cytokines when compared with those seen in BSA-sensitized mice. In BALB/c mice sensitized with Le(x)-BSA or Le(x) mixed with ovalbumin, significantly increased levels of specific IgE and IgG2a antibodies were found concomitant with reduced levels of serum IL-12p70. These effects were attenuated in IL-12-deficient BALB/c mice. Le(x) and an isomer, Le(y), but not other isomers, inhibited the production of LPS-induced IL-12p70, associated with a significant reduction of nuclear NF-kappaB, in bone marrow-derived dendritic cells from BALB/c mice, suggesting that Le(x)-induced suppression of IL-12p70 results in an enhanced T(H)2 response. The addition of mannan, a known ligand for dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin, abrogated the suppressive effect of Le(x) trisaccharides. CONCLUSION: These results provide evidence for a potential role of Le(x) trisaccharides in shaping the immune responses through, at least in part, its suppressive effect on IL-12p70 production. Considering the relative ubiquity of glycoproteins with Le(x) or similar oligosaccharides, including plant-derived (or food-derived) allergens, these findings might have a broad implication. CLINICAL IMPLICATIONS: The adjuvant activity of Le(x) trisaccharides might aid in vaccine design and might be important in determining the allergenicity of proteins containing this or other similar structures.

Genetic variants of the T-cell immunoglobulin mucin 1 but not the T-cell immunoglobulin mucin 3 gene are associated with asthma in an African American population.

BACKGROUND: The T-cell immunoglobulin mucin ( TIM ) proteins and their genetic variants have been suggested to play a role in regulating allergic diseases. OBJECTIVE: Genetic association of the sequence variants for TIM-1 and TIM-3 genes with asthma in an African American population was investigated. METHODS: Both case-control and family-based association analyses were performed for a total of 7 polymorphisms, including 3 single nucleotide polymorphism (SNPs) and 1 insertion/deletion polymorphism in the TIM-1 and 3 SNPs in the TIM-3 genes. The exposure to hepatitis A virus as judged by seropositivity was also examined. RESULTS: In the case-control design, the frequencies of the TT genotype for SNP rs2277025 and the homozygous deletion variant (157delMTTTVP) in the fourth exon of the TIM-1 gene were higher among patients with patients with asthma compared with the controls (odds ratio [OR], 2.779, P = .016; and OR, 3.09, P = .022, respectively). This association was substantiated by haplotype analysis of these and 2 additional SNPs (OR, 2.48; P = .004), and also by family-based tests for the allele and haplotype carrying 157delMTTTVP (P = .009 and P = .048, respectively). Furthermore, this association seems to exist even in the hepatitis A virus-seronegative subjects in our data. None of the 3 variants in TIM-3 genes yielded significant association with either asthma or asthma-related phenotypes. CONCLUSION: Our findings suggest that the genetic variants of the TIM-1 but not the TIM-3 gene contribute to asthma susceptibility in this African-American population.

Interleukin-17F induces pulmonary neutrophilia and amplifies antigen-induced allergic response.

Interleukin (IL)-17F is a recently described human cytokine belonging to the IL-17 gene family, but its in vivo function remains to be determined. To this end, a full-length mouse IL-17F cDNA sequence with a 483-bp coding region sequence was first identified. Pulmonary gene transfer of an IL-17F expression construct (pcDNAmIL-17F) in mice was used to investigate its regulatory role. The results showed first that a significant increase in the number of neutrophils was seen in the bronchoalveolar lavage fluids of IL-17F-transduced mice, concomitant with increased expression of genes encoding C-X-C chemokines and inflammatory cytokines when compared with mock and phosphate-buffered saline control animals. Mucosal transfer of the IL-17F gene in ovalbumin (OVA)-sensitized mice before antigen (Ag) challenge enhanced the levels of Ag-induced pulmonary neutrophilia, but not eosinophilia, goblet cell hyperplasia, and mucin gene expression. However, no significant change in the levels of Th2 cytokine expression was noted. A significant enhancement of ventilatory timing in response to inhaled methacholine was also seen in IL-17F-transduced, Ag-sensitized mice, whereas a small but significant increase was found in IL-17F-transduced, naive mice. These results suggest a role for IL-17F in the induction of neutrophilia in the lungs and in the exacerbation of Ag-induced pulmonary inflammation.

Regulation of TH2 responses by the pulmonary Clara cell secretory 10-kd protein.

BACKGROUND: Pulmonary Clara cell secretory 10-kd protein (CC10) is a steroid-inducible and potentially anti-inflammatory cytokine, but its direct involvement in the regulation of T-cell responses remains unknown. OBJECTIVE: The role of CC10 in the regulation of T(H)2 cytokine expression was investigated. METHODS: The levels of cytokine and GATA-3 expression were determined by ELISA and RT-PCR, respectively. Bronchoalveolar lavage fluid cell counts were also determined by using a standard protocol. CC10 expression in vivo was determined by immunocytochemistry and Western blotting. RESULTS: In vitro, a significant, dose-dependent suppressive effect of CC10 was found on T(H)2 cytokine expression, but not IFN-gamma, in splenocytes of antigen-sensitized mice. A similar suppressive effect was also noted in polarized CD4(+) T(H)2 cells, but not in naive CD4(+) T cells. In contrast, CC10 was able to induce IFN-gamma expression in naive CD4(+) T cells, but not in polarized T(H)1 cells. Furthermore, the suppression of T(H)2 cytokine expression was concomitant with reduction of a critical transcription factor, GATA-3. Of significance was the finding that although no significant change was found in the decay kinetics of T(H)2 cytokine transcripts, a significant decrease in mRNA stability of GATA-3 was seen in CC10-treated cells. In vivo, reconstitution of the CC10 gene in CC10-deficient mice resulted in significantly lower levels of T(H)2 cytokines, concomitant with a decrease in GATA-3 expression, after challenge with Ag compared with those seen in mock-transduced mice, which are associated with reduced levels of pulmonary eosinophilia. CONCLUSION: These results demonstrate, that CC10 plays a direct role in the regulation of T-cell-mediated inflammatory responses.

Sequence variants of the gene encoding chemoattractant receptor expressed on Th2 cells (CRTH2) are associated with asthma and differentially influence mRNA stability.

The gene, CRTH2, encoding a receptor for prostaglandin D(2) (PGD(2)), is located within the peak linkage region for asthma on chromosome (Chr.) 11q reported in African American families. Family-based analysis of asthma and two common SNPs [G1544C and G1651A (rs545659)] in the 3'-untranslated region of CRTH2 showed significant evidence of linkage in the presence of disequilibrium for the 1651G allele (P = 0.003) of SNP rs545659. Haplotype analysis yielded additional evidence of linkage disequilibrium for the 1544G-1651G haplotype (P < 0.001). Population-based case-control analyses were conducted in two independent populations, and demonstrated significant association of the 1544G-1651G haplotype with asthma in an African American population (P = 0.004), and in a population of Chinese children (P < 0.001). Moreover, in the Chinese children the frequency of the 1651G allele in near-fatal asthmatics was significantly higher than mild-to-moderate asthmatics (P = 0.001) and normal controls (P < 0.001). The 1651G allele of SNP re545659 was also associated with a higher degree of bronchial hyperresponsiveness (P < 0.027). Transcriptional pulsing experiments showed that the 1544G-1651G haplotype confers a significantly higher level of reporter mRNA stability, when compared with a non-transmitted haplotype (1544C-1651A), suggesting that the CRTH2 gene on Chr. 11q is a strong candidate gene for asthma.

Evidence for asthma susceptibility genes on chromosome 11 in an African-American population.

Initial genome-wide scan data provided suggestive evidence for linkage of the asthma phenotype in African-American (AA), but not Caucasian, families to chromosome 11q markers (peak at D11S1985; LOD=2). To refine this region, mapping analysis of 91 AA families (51 multiplex families and 40 asthmatic case-parent trios) was performed with an additional 17 markers flanking the initial peak linkage marker. Multipoint analyses of the 51 multiplex families yielded significant evidence of linkage with a peak non-parametric linkage score of 4.38 at marker D11S1337 (map position 68.6 cM). Furthermore, family-based association and transmission disequilibrium tests conducted on all 91 families showed significant evidence of linkage in the presence of disequilibrium for several individual markers in this region. A putative susceptibility locus was estimated to be at map position 70.8 cM with a confidence interval spanning the linkage peak. Evidence from both linkage and association analyses suggest that this region of chromosome 11 contains one or more susceptibility genes for asthma in these AA families.