Breast milk IgA to foods has different epitope specificity than serum IgA-Evidence for entero-mammary link for food-specific IgA?

BACKGROUND: We have previously shown that maternal cow's milk (CM) elimination results in downregulation of CM-specific IgA antibody levels in BM, but not in serum, suggesting that an entero-mammary link may exist for food-specific antibody-secreting cells. OBJECTIVE: We sought to investigate whether food-specific IgA epitope profiles differ intra-individually between mother's serum and BM. We also examined how infants' food epitope-specific IgA develops in early infancy and the relationship of IgA epitope recognition with development of cow's milk allergy (CMA). METHODS: We measured specific IgA to a series of overlapping peptides in major CM allergens (αs1 -, αs2 -, ß- and κ-caseins and ß-lactoglobulin) in paired maternal and infant serum as well as BM samples in 31 mother-infant dyads within the first 15 post-partum months utilizing peptide microarray. RESULTS: There was significant discordance in epitope specificity between BM and maternal sera ranging from only 13% of sample pairs sharing at least one epitope in αs1 -casein to 73% in κ-casein. Epitope-specific IgA was detectable in infants' sera starting at less than 3 months of age. Sera of mothers with a CMA infant had increased binding of epitope-specific IgA to CM proteins compared to those with a non-CMA infant. CONCLUSION & CLINICAL RELEVANCE: These findings support the concept that mother's milk has a distinct antifood antibody repertoire when compared to the antibody repertoire of the peripheral blood. Increased binding of serum epitope-specific IgA to CM in mothers of infants with CMA may reflect inherited systemic immunogenicity of CM proteins in these families, although specific IgA in breast milk was not proportionally up-regulated.

Pathogenesis of IgE-mediated food allergy.

Food allergy is a prevalent disease for which there is no current treatment beyond careful food avoidance. Accidental exposure to foods causes reactions in allergic individuals that can range in severity from mild skin reactions to severe and life-threatening anaphylaxis, and there are no validated tools to predict severity of reactions. A greater understanding of the pathogenesis of food allergy is needed to develop prevention and treatment strategies for food allergy. In the last few years, there have been significant developments in the field of food allergy that have led to new ideas about food allergy prevention, diagnosis, and treatment. This review will discuss these recent advances in the food allergy field as well as identify gaps in our knowledge about the immune mechanisms of allergy and tolerance to foods.

In vivo methods for testing allergenicity show that high hydrostatic pressure hydrolysates of ß-lactoglobulin are immunologically inert.

The major milk allergen ß-lactoglobulin (ß-LG) exhibits an enhanced susceptibility to proteolysis under high hydrostatic pressure and this may be an efficient method to produce hypoallergenic hydrolysates. The aim of this work was to evaluate the in vivo allergenicity of 3 ß-LG hydrolysates produced under atmospheric pressure or high-pressure conditions. Hydrolysates were chosen based on previous experiments that showed that they provide a complete removal of intact ß-LG but differed in vitro IgE-binding properties that could be traced to the peptide pattern. The ability to trigger systemic anaphylaxis was assessed using C3H/HeJ mice orally sensitized to ß-LG. Outcome measures included symptom score, body temperature, serum mouse mast cell protease 1 (mMCP-1), and quantification of circulating basophils. Mast cell degranulation in vivo was assessed by passive cutaneous anaphylaxis. The 3 tested hydrolysates showed an abrogated allergenicity as revealed by the absence of anaphylactic symptoms and a decrease in body temperature. We demonstrated that the peptides present in the hydrolysates had lost their ability to cross-link 2 human IgE antibodies to induce mast cell degranulation, thus indicating that most of the peptides formed retain just one relevant IgE-binding epitope. The orally sensitized mouse model is a useful tool to address the in vivo allergenicity of novel milk formulas and demonstrates the safety of hydrolysates produced under high-pressure conditions.

Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer's patches.

BACKGROUND: The underlying mechanisms responsible for allergic sensitization to food proteins remain elusive. To investigate the intrinsic properties (as well as the effect of pasteurization) of the milk proteins alpha-lactalbumin, beta-lactoglobulin and casein that promote the induction of milk allergy. METHODS: Alteration of structure and immune-reactivity of native and pasteurized proteins was assessed by gel filtration and ELISA. Uptake of these proteins was compared in vitro and in vivo. The biological effect was assessed by orally sensitizing C3H/HeJ mice with milk proteins followed by a graded oral challenge. Required dose to induce anaphylaxis, symptoms and mean body temperature was recorded. Antigen-specific antibodies and cytokine production by splenocytes were analyzed. RESULTS: Soluble beta-lactoglobulin and alpha-lactalbumin but not insoluble casein were readily transcytosed through enterocytes in vitro and in vivo. Pasteurization caused aggregation of beta-lactoglobulin and alpha-lactalbumin inhibiting uptake by intestinal epithelial cells in vitro and in vivo. Furthermore, aggregation redirected uptake to Peyer's patches, which promoted significantly higher Th2-associated antibody and cytokine production in mice than their native counterparts. Despite this only the soluble forms of beta-lactoglobulin and alpha-lactalbumin elicited anaphylaxis (following priming) when allergens were administered orally. Aggregated beta-lactoglobulin and alpha-lactalbumin as well as casein required systemic administration to induce anaphylaxis. CONCLUSIONS: These results indicate that triggering of an anaphylactic response requires two phases (1) sensitization by aggregates through Peyer's patches and (2) efficient transfer of soluble protein across the epithelial barrier. As the majority of common food allergens tend to form aggregates, this may be of clinical importance.

Enhanced intestinal transepithelial antigen transport in allergic rats is mediated by IgE and CD23 (FcepsilonRII).

We previously reported that active sensitization of rats resulted in the appearance of a unique system for rapid and specific antigen uptake across intestinal epithelial cells. The current studies used rats sensitized to horseradish peroxidase (HRP) to define the essential components of this antigen transport system. Sensitization of rats to HRP stimulated increased HRP uptake into enterocytes (significantly larger area of HRP-containing endosomes) and more rapid transcellular transport compared with rats sensitized to an irrelevant protein or naive control rats. Whole serum but not IgE-depleted serum from sensitized rats was able to transfer the enhanced antigen transport phenomenon. Immunohistochemistry demonstrated that sensitization induced expression of CD23, the low-affinity IgE receptor (FcepsilonRII), on epithelial cells. The number of immunogold-labeled CD23 receptors on the enterocyte microvillous membrane was significantly increased in sensitized rats and was subsequently reduced after antigen challenge when CD23 and HRP were localized within the same endosomes. Finally, pretreatment of tissues with luminally added anti-CD23 antibody significantly inhibited both antigen transport and the hypersensitivity reaction. Our results provide evidence that IgE antibodies bound to low-affinity receptors on epithelial cells are responsible for the specific and rapid nature of this novel antigen transport system.

Immune-epithelial interactions in host defense.

Over the past 15 years, it has become very clear that the immune system can have profound effects on epithelial function. Acute immune-mediated changes in epithelial physiology are beneficial to host defense against enteric pathogens. For example, ion secretion washes out noxious luminal contents and increased permeability allows phagocytic cells and antibodies to enter the gut lumen. However, ongoing immune activation results in chronic effects that may be pathophysiologic. Responses are mediated by soluble immune mediators that act directly on the epithelium, or indirectly via nerves that also serve to amplify the epithelial response. Here, we will review some of the recent advances that have been made in the field of immunophysiology. The effect of mast cells on transport functions of the epithelium will be reviewed, with emphasis on the consequence of interactions between mast cells and nerves. The use of in vitro coculture systems has recently provided considerable information on the effects of neutrophils, eosinophils, monocytes, and lymphocytes on epithelial functions; the contribution of each immunocyte will be highlighted. Finally, we will describe evidence for the active participation of the epithelium in mucosal immune activation, including pathogen or cytokine induced epithelial cytokine synthesis or secretion and adhesion molecule expression.

Effect of psychoneural factors on intestinal epithelial function.

Stress has been associated with abnormal gastrointestinal function, including diarrhea and abdominal pain, and stress-associated gastric ulceration has frequently been documented. Stress can also exacerbate ongoing pathophysiology and often precedes relapses in patients with inflammatory bowel disease or irritable bowel syndrome. The relatively new field of psychoneuroimmunology is involved with the elucidation of mechanisms that explain the link between the central nervous system and immune-mediated pathophysiology. Recent progress examining the interaction among the nervous system, the immune system and the epithelium of the intestine is discussed, and the evidence for central nervous system control of this interaction is examined.

TNFα-dependent development of lymphoid tissue in the absence of RORγt⁺ lymphoid tissue inducer cells.

Lymphoid tissue often forms within sites of chronic inflammation. Here we report that expression of the proinflammatory cytokine tumor necrosis factor α (TNFα) drives development of lymphoid tissue in the intestine. Formation of this ectopic lymphoid tissue was not dependent on the presence of canonical RORgt(+) lymphoid tissue-inducer (LTi) cells, because animals expressing increased levels of TNFα but lacking RORgt(+) LTi cells (TNF/Rorc(gt)(-/-) mice) developed lymphoid tissue in inflamed areas. Unexpectedly, such animals developed several lymph nodes (LNs) that were structurally and functionally similar to those of wild-type animals. TNFα production by F4/80(+) myeloid cells present within the anlagen was important for the activation of stromal cells during the late stages of embryogenesis and for the activation of an organogenic program that allowed the development of LNs. Our results show that lymphoid tissue organogenesis can occur in the absence of LTi cells and suggest that interactions between TNFα-expressing myeloid cells and stromal cells have an important role in secondary lymphoid organ formation.

Mouse and human Notch-1 regulate mucosal immune responses.

The Notch-1 signaling pathway is responsible for homeostatic tight junction expression in vitro, and promotes barrier function in vivo in the RAG1-adoptive transfer model of colitis. In this study, we sought to determine the role of colonic Notch-1 in the lymphoepithelial crosstalk in health and disease. We utilized in vivo and in vitro knockdown to target the expression of Notch-1. We identified that epithelial Notch-1 is required for appropriate activation of intestinal epithelial cells at steady state and upon inflammatory stimulus. Notch-1 expression modulates mucosal chemokine and cytokine secretion, and FoxP3 and effector T-cell responses. We showed that epithelial Notch-1 controls the immune function of the epithelium through crosstalk with the nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathways that, in turn, elicits T-cell responses. Overall, epithelial Notch-1 bridges innate and adaptive immunity in the gut. Our findings highlight an indispensable role for Notch-1-mediated signaling in the intricate epithelial-immune crosstalk, and validate that epithelial Notch-1 is necessary and sufficient to support protective epithelial proinflammatory responses.

Reduced severity of peanut-induced anaphylaxis in TLR9-deficient mice is associated with selective defects in humoral immunity.

Signaling through the innate immune system can promote or suppress allergic sensitization. Toll-like receptor 9 (TLR9) has modulatory effects on the mucosal immune system, and we hypothesized that TLR9 would influence susceptibility to allergic sensitization to foods. We observed that TLR9-/- mice were resistant to peanut-induced anaphylaxis. This was associated with a significant impairment in total immunoglobulin E (IgE) and peanut-specific IgE and IgA, but not IgG1 or Th2 cytokine production. TLR9-/- mice had reduced development of Peyer's patches, but resistance to sensitization was not restricted to oral routes. Rag1-deficient mice were reconstituted with TLR9+/+ or -/- B cells plus CD4+ T cells. TLR9-/- B cells regained the ability to produce IgE in the presence of a wild-type environment. Our results demonstrate that TLR9 on an unknown cell type is required for the development of IgE-producing B cells, and we conclude that TLR9 signaling indirectly shapes the immune response for optimal IgE production.

Immunophysiology of experimental food allergy.

Animal models of food allergy have been used to identify mechanisms involved in the development of sensitization to food proteins as well as immunologic mechanisms of adverse reactions to allergen reexposure. To counteract the normal tolerant responses to antigen generated in the gastrointestinal tract, investigators have used mucosal adjuvants or manipulated the mucosal barrier, taken advantage of endogenous adjuvanticity of some food allergens, or bypassed the oral route and sensitized through the skin. Site of antigen uptake in the gastrointestinal tract is a critical factor in both sensitization and anaphylaxis, and antigen uptake can be facilitated by immunoglobulin-E (IgE)-antigen complexes binding to CD23 on the epithelial cell surface. Studies on systemic anaphylaxis or local gastrointestinal manifestations of food allergy in mice have highlighted the contribution of IgE, mast cells, and pathogenic Th2 lymphocytes in experimental food allergy.

Role of TLR4 in allergic sensitization to food proteins in mice.

Allergic sensitization to food proteins and other allergens is increasing in prevalence. One hypothesis for this increase is that the decreased rate of infections or exposure to microbial products leaves the immune system susceptible to inappropriate reactivity to innocuous antigens through the lack of development of regulatory cells. We hypothesized that constitutive Toll-like receptor (TLR)4 signaling (presumably via the commensal flora) could inhibit the development of allergic sensitization to food proteins. We tested this hypothesis by sensitizing TLR4+ and TLR4- mice on two genetic backgrounds, C3H and BALB/c, to two common food allergens [beta-lactoglobulin (betaLG) and peanut (PN)]. B-cell responses were not significantly influenced by TLR4 status. T-cell responses were Th2 skewed in TLR4-deficient C3H mice compared with TLR4 sufficient C3H mice, but this pattern of Th2 skewing was not observed in TLR4-deficient mice on a BALB/c background. In anaphylaxis-susceptible C3H mice, TLR4 deficiency was associated with increased severity of anaphylaxis to PN, and decreased severity of anaphylaxis to betaLG. In anaphylaxis-resistant BALB/c mice, TLR4 deficiency was not sufficient to render mice susceptible to PN-induced anaphylaxis. We conclude that although TLR4 status can influence T-cell responses and anaphylaxis severity, the nature of the influence is highly antigen- and strain-dependent.

Neutrophil-independence of the initiation of colonic injury. Comparison of results from three models of experimental colitis in the rat.

Although the role of neutrophils in the pathogenesis of several forms of gastrointestinal injury has been well demonstrated, their role in the development of experimental colonic injury is less clear. To examine whether neutrophils play a role in the development of experimental colitides, the effects of a sustained neutropenia on multiple indices of colonic injury in rats was examined 24 hr following the initiation of colitis with the intrarectal application of acetic acid, trinitrobenzene sulfonic acid (TNBS)-ethanol, or the potent proinflammatory agent, phorbol-12-myristate-13-acetate (PMA). In comparison to animals with normal neutrophil counts and colitis induced by any of the three agents, no attenuation in macroscopic damage or histopathologic injury was observed in neutropenic animals exhibiting a greater than 95% reduction in circulating neutrophils and 85% reduction in tissue-associated myeloperoxidase activity. Although the tissue edema associated with acetic acid or PMA-induced colitis was not reduced by neutropenia, the colonic edema associated with TNBS colitis was attenuated by prior neutrophil depletion with anti-neutrophil antiserum. Despite our initial hypothesis that neutrophils played a key role in the genesis of experimental colitis (especially that induced by PMA), the results demonstrated that these cells are not essential for the development of the major pathological features of colitis induced by this agent, acetic acid, or TNBS. Although these results support the proposal that in these models of colitis, inflammation develops secondary to injury (rather than the converse), further studies will be necessary to elucidate the role of inflammatory cells other than neutrophils in the genesis of experimental colitides.

Phorbol myristate acetate ex vivo model of enhanced colonic epithelial permeability. Reactive oxygen metabolite and protease independence.

The initiating mechanisms involved in colonic injury are currently unknown. The goal of the current study was to examine the role of the inflammatory mediators reactive oxygen metabolites and proteases in an ex vivo model of selective epithelial permeability. Rats were prepared with exteriorized colonic chambers to which the protein kinase C (PKC) activator phorbol myristate acetate (PMA) was added in doses ranging from 5 to 800 micrograms. PMA caused a dose-dependent transient increase in epithelial permeability, but had no significant effect on microvascular permeability. There was no accumulation of neutrophils and no apparent histological changes. PMA acts via a PKC-dependent mechanism, as assessed using the PKC-inactive phorbol analog 4 alpha-phorbol didecanoate, and the response is tachyphylactic. The mechanism is independent of reactive oxygen metabolites and proteases, as shown by the lack of effect of the free radical scavengers superoxide dismutase and catalase and the general serine protease inhibitor soybean trypsin inhibitor. The classic inflammatory process does not appear to be involved in the PMA-induced epithelial permeability changes. This finding suggests that noninflammatory mechanisms may regulate the increased epithelial permeability induced by PMA. Further study to elucidate these mechanisms is of importance for understanding both normal gastrointestinal physiology and initiation of pathology.

Enhanced antigen transport across rat tracheal epithelium induced by sensitization and mast cell activation.

Ag challenge to the apical surface of tracheal epithelium results in a rapid ion secretory response due to the activation of mast cells. The aim of this study was to examine the impact of sensitization and specific Ag challenge on the timing, route, and quantity of Ag transported across tracheal epithelium. After sensitization of rats to a model protein, HRP, tracheal tissues were excised and mounted in Ussing chambers. Tracheas from HRP-sensitized rats, but not naive or OVA-sensitized rats, responded to apical HRP challenge with a rise in short-circuit current (beginning at approximately 2 min). Photomicrographs of tissues fixed at 2 min showed that initial transepithelial HRP transport occurred via endosomes and was significantly enhanced in HRP-sensitized rats compared with both control groups. In addition, nonciliated cells, the proportion of which increased after sensitization, contained significantly more HRP than ciliated cells. The hypersensitivity response occurred only in HRP-sensitized and challenged rats and was associated with increased conductance of tracheal epithelium and overall flux of HRP across the tissue. This increased flux of Ag and elevated conductance was not observed in mast cell-deficient Ws/Ws rats. Photomicrographs of tissues fixed 90 min after challenge also showed HRP in the paracellular spaces between adjacent epithelial cells. We conclude that sensitization increases uptake of specific Ag initially via an endosomal transcellular pathway across tracheal epithelium and that, after the hypersensitivity reaction, mast cell-dependent recruitment of the paracellular pathway further augments Ag influx into airway tissue.

Pertussis adjuvant prolongs intestinal hypersensitivity.

BACKGROUND: Immediate hypersensitivity reactions are a hallmark of allergic disease, and result in the clinical features of food allergy, hayfever, and atopic asthma. The mechanism by which an individual becomes sensitized to an ingested or airborne allergen is not clear, however exposure to bacteria or bacterial products that act as adjuvants may be a contributing factor. The purpose of this study was to examine the role of pertussis toxin (PT) in inducing intestinal hypersensitivity reactions, particularly the ability of the adjuvant to prolong the sensitization. METHODS: Rats were sensitized to ovalbumin (OA) by injection of OA alone or with 50 ng PT. Secretory responses to OA challenge and nerve stimulation were assessed in jejunal tissues mounted in Ussing chambers. RESULTS: Jejunal segments from rats sensitized to OA alone responded to antigen challenge with ion secretion, but sensitization was transient in that specific IgE titers and responses to luminal antigen disappeared by 14 days. In contrast, co-administration of 50 ng PT with OA resulted in long-lasting sensitization. Secretory responses to both luminal and serosal OA challenge were present 8 months after primary immunization. Enhanced secretory responses to nerve stimulation, increased mucosal mast cell numbers, as well as elevated IgE titers were also induced and may have contributed to the overall responsiveness of the intestine to antigen challenge. CONCLUSIONS: Our findings indicate nanogram quantities of PT, when administered with a food protein, result in long-term sensitization to the antigen, and altered intestinal neuroimmune function. These data suggest that exposure to bacterial pathogens may prolong the normally transient immune responsiveness to inert food antigens.

Effects of neuropeptide Y and substance P on antigen-induced ion secretion in rat jejunum.

We previously described a model of intestinal hypersensitivity in which isolated gut segments from sensitized rats demonstrated a rapid epithelial secretory response to luminal antigen that was mediated by mucosal mast cells and capsaicin-sensitive nerves. In this study, we examined the ability of the inhibitory neuropeptide, neuropeptide Y (NPY), to diminish the antigen-induced secretory response. Rats were sensitized to egg albumin (EA), and 12-14 days later, jejunal tissue was excised and mounted in Ussing chambers. NPY inhibited the short-circuit current (Isc) increase and Cl- secretion evoked by addition of EA to the luminal side of the tissue; neural blockade with tetrodotoxin (TTX) had a similar inhibitory effect. In contrast, NPY was much less effective, and TTX was completely ineffective, on the response to serosal antigen. Additional experiments examined the cell target for NPY action. NPY and TTX almost abolished the Isc response to electrical transmural stimulation of enteric nerves, suggesting a possible neural site of action. In addition, NPY significantly reduced baseline Isc; this inhibition involved both TTX-dependent and TTX-independent components. Because nerves were previously shown to facilitate antigen uptake and substance P was implicated in the response to only luminal antigen, we postulated that NPY was inhibiting nerves that facilitate antigen transport from the lumen to effector cells in the lamina propria. We therefore examined the effect of exogenous substance P added after NPY inhibition. Substance P restored the luminal antigen-induced secretory response to pretreatment values. We conclude that the neuropeptides play a significant role in immunophysiology by acting at neural and epithelial sites in the intestinal mucosa.

Role for IL-4 in macromolecular transport across human intestinal epithelium.

Increased epithelial permeability is associated with intestinal inflammation, but there is little information on factors that regulate barrier function in the absence of or before inflammation. We examined if interleukin (IL)-4, or serum from atopic individuals, could alter the barrier function of human colonic epithelial (T84) monolayers to antigenic-sized macromolecules. IL-4 and atopic serum significantly decreased T84 monolayer resistance and increased transepithelial horseradish peroxidase (HRP) transport. Bidirectional transport studies demonstrated that IL-4 selectively enhanced apical-to-basal movement of HRP. HRP transport induced by IL-4 was inhibited by cold (4 degrees C) and the tyrosine kinase inhibitor genistein, but not the protein kinase C inhibitor staurosporine. Electron microscopic analysis demonstrated that both transcellular and paracellular pathways were affected. Anti-IL-4 antibodies abolished the increase in HRP transport in response to both IL-4 and serum. We speculate that enhanced production of IL-4 in allergic conditions may be a predisposing factor to inflammation by allowing uptake of luminal antigens that gain access to the mucosal immune system.

Mucosal pathophysiology and inflammatory changes in the late phase of the intestinal allergic reaction in the rat.

Relatively little information exists concerning the late phase of the allergic reaction in the gastrointestinal tract. Here, we characterized jejunal mucosal pathophysiology and inflammation after oral antigen challenge of sensitized rats, and examined the role of mast cells in events after challenge. Sprague-Dawley rats, mast cell-deficient (Ws/Ws), and +/+ control rats were sensitized to horseradish peroxidase, and challenged intragastrically with antigen 14 days later. Jejunal segments were obtained at 0.5 to 72 hours after challenge for functional assessment in Ussing chambers and for morphological assessment by light and electron microscopy. Intestine from sensitized Sprague-Dawley rats demonstrated enhanced ion secretion and permeability at all times after challenge. Electron microscopy revealed abnormal mitochondria within enterocytes and disruption of the epithelial basement membrane associated with influx into the mucosa of mast cells, eosinophils, neutrophils, and mononuclear cells. Many inflammatory cells appeared activated. In contrast, antigen-challenged Ws/Ws rats demonstrated no functional changes or inflammatory cell infiltrate. We conclude that oral antigen challenge of sensitized rats induces sustained epithelial dysfunction. Mast cells mediate both epithelial pathophysiology and recruitment of additional inflammatory cells that may contribute to persistent pathophysiology and symptoms.

Regulated production of the T helper 2-type T-cell chemoattractant TARC by human bronchial epithelial cells in vitro and in human lung xenografts.

The chemokine TARC is a ligand for the chemokine receptor CCR4 expressed on T helper (Th)2-type CD4 T cells. Allergic airway inflammation is characterized by a local increase in cells secreting Th2-type cytokines. We hypothesized that bronchial epithelial cells may be a source of chemokines known to chemoattract Th2 cells. Regulated TARC expression was studied using normal human bronchial epithelial cells and a human lung xenograft model. TARC expression was increased in normal human bronchial epithelial cells in response to tumor necrosis factor-alpha stimulation, and further upregulation of TARC was observed with interferon (IFN)-gamma but not interleukin (IL)-4 costimulation. TARC functions as a nuclear factor (NF)-kappa B target gene, as shown by the abrogation of TARC expression in response to proinflammatory stimuli when NF-kappa B activation is inhibited. In an in vivo model, minimal constitutive TARC expression was observed in human lung xenografts. Consistent with our findings in vitro, TARC messenger RNA (mRNA) expression was upregulated in the xenografts in response to IL-1, and costimulation with IFN-gamma but not IL-4 further increased TARC mRNA and protein expression. In addition, bronchoalveolar lavage fluid from asthmatic subjects after allergen challenge contained significantly increased levels of TARC, suggesting that TARC production by bronchial epithelial cells may play a role in the pathogenesis of allergic asthma.