Airway generation-specific differences in the spatial distribution of immune cells and cytokines in allergen-challenged rhesus monkeys.

BACKGROUND: Accumulation of immune cell populations and their cytokine products within tracheobronchial airways contributes to the pathogenesis of allergic asthma. It has been postulated that peripheral regions of the lung play a more significant role than proximal airways with regard to inflammatory events and airflow obstruction. OBJECTIVE: To determine whether immune cell populations and associated cytokines are uniformly distributed throughout the conducting airway tree in a non-human primate model of allergic asthma. METHODS: We used a stereologic approach with a stratified sampling scheme to measure the volume density of immune cells within the epithelium and interstitium of trachea and 4-5 intrapulmonary airway generations from house dust mite (HDM) (Dermatophagoides farinae)-challenged adult monkeys. In conjunction with immune cell distribution profiles, mRNA levels for 21 cytokines/chemokines and three chemokine receptors were evaluated at four different airway generations from microdissected lungs. RESULTS: In HDM-challenged monkeys, the volume of CD1a+ dendritic cells, CD4+ T helper lymphocytes, CD25+ cells, IgE+ cells, eosinophils, and proliferating cells were significantly increased within airways. All five immune cell types accumulated within airways in unique patterns of distribution, suggesting compartmentalized responses with regard to trafficking. Although cytokine mRNA levels were elevated throughout the conducting airway tree of HDM-challenged animals, the distal airways (terminal and respiratory bronchioles) exhibited the most pronounced up-regulation. CONCLUSION: These findings demonstrate that key effector immune cell populations and cytokines associated with asthma differentially accumulate within distinct regions and compartments of tracheobronchial airways from allergen-challenged primates.

Modulation of inhaled antigen-induced IgE tolerance by ongoing Th2 responses in the lung.

The normal response to inhaled Ag is the absence of Ag-specific IgE and cytokine production to later Ag challenges. Although the mechanism of this aerosol-induced IgE tolerance is not completely understood, it may prevent sensitization to inhaled Ags, which could otherwise lead to allergy and asthma. We examined the consequences of ongoing Th1 and Th2 responses in the lungs of mice during OVA inhalation to mimic conditions that may subvert tolerance and lead to sensitization. We found that concurrent, secondary Th2 lung responses to keyhole limpet hemocyanin or primary responses to Nippostrongylus larvae or Asperigillus fumagatus extract prevented establishment of IgE tolerance to aerosolized OVA. Intranasal rIL-4 given before OVA aerosolization also prevented establishment of tolerance, whereas concurrent Th1 responses to influenza virus or Mycobacterium bovis bacillus Calmette-Guérin had no effect. However, once established, aerosol tolerance to OVA could not be completely broken by OVA rechallenge concurrent with a secondary Th2 response to keyhole limpet hemocyanin or A. fumagatus extract, or by intranasal rIL-4. These data suggest that the immune status of the lung of an individual may profoundly influence the initial response to inhaled Ag, and that aerosol-induced IgE tolerance may not be appropriately established in individuals undergoing concurrent, Th2-mediated responses to Ags or pathogens.

IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo.

We have characterized a cytokine produced by Th2 cells, designated as IL-25. Infusion of mice with IL-25 induced IL-4, IL-5, and IL-13 gene expression. The induction of these cytokines resulted in Th2-like responses marked by increased serum IgE, IgG(1), and IgA levels, blood eosinophilia, and pathological changes in the lungs and digestive tract that included eosinophilic infiltrates, increased mucus production, and epithelial cell hyperplasia/hypertrophy. In addition, our studies show that IL-25 induces Th2-type cytokine production by accessory cells that are MHC class II(high), CD11c(dull), and lineage(-). These results suggest that IL-25, derived from Th2 T cells, is capable of amplifying allergic type inflammatory responses by its actions on other cell types.

Anti-interleukin 10 receptor monoclonal antibody is an adjuvant for T helper cell type 1 responses to soluble antigen only in the presence of lipopolysaccharide.

Soluble foreign antigen usually leads to a transient clonal expansion of antigen-specific T cells followed by the deletion and/or functional inactivation of the cells. As interleukin (IL)-10 is a key immunoregulatory cytokine, we questioned whether neutralization of IL-10 during priming with soluble antigen could prime for a subsequent T helper cell type 1 (Th1) effector recall response. By using an adoptive transfer model to track the fate of antigen-specific T cell receptor (TCR)-transgenic CD4(+) T cells, we show that administration of soluble ovalbumin (OVA) protein, but not OVA(323-339) peptide antigen, together with an anti-IL-10 receptor (R) mAb led to the enhancement of a Th1 response upon rechallenge. Lipopolysaccharide (LPS) present in the protein was necessary for priming for Th1 recall responses in the presence of anti-IL-10R mAb, as removal of LPS abrogated this effect. Moreover, addition of LPS to the peptide did not itself allow priming for recall Th1 effector responses unless endogenous levels of IL-10 were neutralized with an anti-IL-10R mAb. A significant increase in OVA-specific IgG1 and IgG2a isotypes was observed when the protein antigen was administered with anti-IL-10R mAb; however, this was not the case with peptide antigen administered together with anti-IL-10R and LPS. Our data, showing that LPS receptor signaling and neutralization of endogenous immunosuppressive cytokines is essential for Th1 priming, has important implications for the design of relevant vaccines for effective in vivo immunotherapy.

T regulatory cells 1 inhibit a Th2-specific response in vivo.

We recently described a new population of CD4(+) regulatory T cells (Tr1) that inhibits proliferative responses of bystander T cells and prevents colitis induction in vivo through the secretion of IL-10. IL-10, which had been primarily described as a Th2-specific cytokine inhibiting Th1 responses, has displayed in several models a more general immune suppression on both types of effector T cell responses. Using an immediate hypersensitivity model in which BALB/c mice immunized with OVA (alum) normally generate Th2-dominated responses, we examined the ability of OVA-specific Tr1 T cell clones to inhibit OVA-specific cytokines and Ab responses. In contrast to Th2 or Th1 T cell clones, transfer of Tr1 T cell clones coincident with OVA immunization inhibited Ag-specific serum IgE responses, whereas IgG1 and IgG2a synthesis were not affected. This specific inhibition was mediated in part through IL-10 secretion as anti-IL-10 receptor Abs treatment reverted the inhibitory effect of Tr1 T cell clones. Although specifically targeted to IgE responses, Tr1 clones' inhibitory effects were more profound as they affected Ag-specific Th2 cell priming both in term of proliferative responses and cytokine secretion. These results suggest that regulatory T cells may play a fundamental role in maintaining the balance of the immune system to prevent allergic disorders.

Interferon gamma induction during oral tolerance reduces T-cell migration to sites of inflammation.

BACKGROUND & AIMS: Previous data suggest that oral antigen induces interferon (IFN)-gamma production in intestinal T cells. However, oral tolerance is associated with decreased production of IFN-gamma by T cells after antigen sensitization. The aim of this study was to examine the role of IFN-gamma in oral tolerance. METHODS: Oral tolerance was examined in BALB/c mice after the adoptive transfer of T cells from chicken ovalbumin (OVA(323-339))-specific, DO11.10 x RAG-1(-/-) T-cell receptor transgenic mice. RESULTS: OVA feeding induced systemic tolerance of delayed-type hypersensitivity (DTH) and antibody responses. OVA feeding up-regulated IFN-gamma production by transgenic T cells in Peyer's patch and mesenteric lymph node but not splenic tissues. Treatment of OVA-fed mice with neutralizing monoclonal antibody to IFN-gamma prevented tolerance of DTH responses. Analysis of transgenic T-cell numbers in DTH sites by immunohistochemical staining suggested that induction of IFN-gamma by oral antigen decreased accumulation of transgenic T cells in cutaneous sites of antigen injection. IFN-gamma-deficient or wild-type DO11.10 and BALB/c mice were used to show that IFN-gamma production by donor transgenic T cells was critical for oral tolerance. CONCLUSIONS: These data suggest that the induction of IFN-gamma by oral antigen contributes to systemic tolerance by decreasing migration of T cells to peripheral sites of inflammation.

The differentiated state of intestinal lamina propria CD4+ T cells results in altered cytokine production, activation threshold, and costimulatory requirements.

Intestinal lamina propria (LP) CD4+ T cells are memory-like effector cells that proliferate at relatively low levels and require high levels of TCR signaling and costimulation for full activation in vitro. To study LP CD4+ T cell functional potential we used DO11.10 TCR transgenic (Tg) mice specific for the class II MHC-restricted OVA323-339 peptide and nontransgenic BALB/c mice. Activation of LP Tg+ T cells with Ag using mucosal explants induced high levels of IL-2, IL-4, and IFN-gamma. Culturing isolated LP cells with IL-12 enhanced IFN-gamma production and down-regulated IL-4 and IL-2, whereas addition of IL-4 maintained IL-4 production without inhibiting IFN-gamma production. Systemic administration of relatively high dose (HD; 100 nM) OVA323-339 peptide induced similar levels of bromodeoxyuridine (BrdU) incorporation by LP and splenic Tg+ T cells in vivo, whereas low dose (LD; 4.5 nM) peptide injections induced 4-fold greater levels of BrdU incorporation for LP compared with splenic Tg+ T cells. Coadministration of CTLA-4Ig reduced BrdU incorporation for splenic cells by 70% with HD and LD stimulation, but had little effect on LP responses to HD stimulation. Results of in vivo studies were confirmed in nontransgenic BALB/c mice using HD (200 microg) and LD (10 microg) anti-CD3 mAb+/- CTLA-4Ig. These results suggest that LP T cells are differentiated effector cells that respond at high levels when activated with relatively low levels of Ag- and B7-mediated costimulation in vivo. The reduced activation threshold of LP T cells may facilitate responses to low levels of Ag derived from mucosal pathogens.

Lungkine, a novel CXC chemokine, specifically expressed by lung bronchoepithelial cells.

We describe a novel mouse CXC chemokine that is selectively expressed in lung epithelial cells and up-regulated in various lung inflammation models. Although this chemokine clusters with other ELR-CXC chemokines, none of them can confidently be assigned to be its human homologue based on sequence identity. In addition, the highly restricted mRNA tissue distribution of this chemokine differentiates it from all previously described chemokines: Lungkine could not be detected in any of the 70 cDNA libraries analyzed corresponding to specific murine cell populations and tissues. High levels of Lungkine mRNA were specifically detected in the lung and at lower levels in fetal lung tissue by Northern blot and in situ hybridization, suggesting a potential role for this chemokine during lung development. Moreover, Lungkine protein is secreted into the airway spaces and induces the in vitro and in vivo migration of neutrophils, suggesting that it is involved in lung-specific neutrophil trafficking. Using fluorescent in situ hybridization, we show that Lungkine maps to mouse chromosome 5.

Differential CC chemokine-induced enhancement of T helper cell cytokine production.

Chemokines are a family of small m.w. cytokines that induce chemotaxis and chemokinesis of leukocytes. These molecules are ligands for seven-transmembrane, Gi protein-linked receptors that induce a signaling cascade in human T cells and provide costimulation for T cell activation, in addition to participating in transendothelial migration of leukocytes. To address the role of chemokines in the regulation of Th cell cytokine production, we utilized an OVA-specific TCR transgenic (Tg+) model. Cells stimulated through the TCR and incubated in the presence of macrophage inflammatory protein-1alpha (MIP-1alpha) showed enhanced IFN-gamma production, whereas cells incubated in the presence of monocyte chemotactic protein-1 (MCP-1) showed enhanced IL-4 production. Similar results were obtained whether TCR Tg+ T cells were stimulated with anti-CD3 mAb or OVA peptide. Primary stimulation of T cells in the presence of chemokines, followed by secondary stimulation and tertiary stimulation with anti-TCR clonotype mAb alone (no exogenous chemokines), revealed an enhanced IFN-gamma production for MIP-1alpha stimulation and IL-4 production for MCP-1 stimulation. Naive Tg+ T cells, obtained from Tg+ mice crossed to RAG-1-deficient mice, showed enhanced IFN-gamma production when incubated with MIP-1alpha and enhanced IL-4 production when incubated with MCP-1. These results suggest CC chemokines play a role in regulating naive Th cell cytokine production, in addition to regulating leukocyte trafficking.

Functional differentiation of T cells in the intestine of T cell receptor transgenic mice.

The intestinal lamina propria (LP) is a major effector site of the mucosal immune system where antigen-specific and antigen-nonspecific factors shape the functional responses of CD4+ T helper cells. To study the functional differentiation of LP T helper cells we utilized DO11.10 T cell receptor (TCR) transgenic (Tg) mice that expressed a clonotypic TCR specific for a class II major histocompatibility complex-restricted peptide of chicken ovalbumin. The majority of cells expressing Tg TCR (Tg+) in peripheral lymphoid tissue expressed naive surface phenotypes whereas nearly all Tg+ T cells in the intestinal LP expressed an activated/ memory-like phenotype. Flow cytometric analysis of Tg+ T cell populations revealed that a small proportion of cells in peripheral lymphoid tissue but nearly all cells in the LP expressed dual (Tg plus non-Tg) TCRs. In Tg x recombinase-activating-gene-1-deficient (Tg x RAG-1(-/-)) mice, splenic and LP T cells expressed naive surface phenotypes and produced cytokines equivalent to naive splenic cells from Tg x RAG-1(+/+) mice. In contrast, Tg LP cells from Tg x RAG-1(+/+) mice produced 35-fold greater levels of interferon-gamma and 5-fold greater levels of interleukin 4 compared with naive splenic cells. These findings suggested that activation of Tg+ T cells through endogenous non-Tg TCR had promoted the localization and differentiation of memory-like effector T helper cells in the intestine.

Abrogation of a mitotic checkpoint by E2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p53 tumor suppressor protein.

Human papillomavirus (HPV) E2 and E1 proteins are required for the replication of viral genomes in vivo. We have examined the effects of increasing the level of E2 on viral and cellular replication using recombinant adenoviruses. Infection of cells which maintain HPV 31 DNA episomally with E2 recombinant adenoviruses resulted in a 5-fold increase in genome copy number as well as an S phase arrest allowing for the continued replication of cellular DNA. Similar effects on cell cycle progression were seen following infection of normal human foreskin keratinocytes, the natural host cell. The DNA content of these cells increased beyond 4N indicating that multiple rounds of replication had occurred without an intervening mitotic event. In addition, increased cyclin A and E associated kinase activity was observed, while no change was detected in cyclin B associated kinase activity or in the activation state of cdc2 kinase. Interestingly, the levels of the p53 tumor suppresser protein were dramatically reduced through a post-transcriptional mechanism following infection. These data suggest a role for E2 in regulating viral and cellular replication by abrogation of a mitotic checkpoint, which is, at least in part, controlled by p53.

IFN-gamma-activated primary murine astrocytes express B7 costimulatory molecules and prime naive antigen-specific T cells.

Astrocytes may serve as effectual APCs for T cell-mediated immune responses to myelin components during multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Although astrocytes have been reported not to constitutively express MHC class II molecules, expression is up-regulated during active EAE and by in vitro incubation with IFN-gamma. Previous studies have reported that cytokine-activated astrocytes are able to activate Ag-specific previously activated T cells, but not naive alloreactive T cells. In the current study, we show that a subset of primary murine astrocytes constitutively expresses B7-2 molecules, as determined by FACS and PCR analyses, and up-regulates surface expression and mRNA levels of both B7-2 and B7-1 upon IFN-gamma stimulation. In contrast to earlier reports, we found that both untreated and IFN-gamma-treated astrocytes were able to stimulate proliferation of previously activated OVA-specific Th1 cells. In contrast, only IFN-gamma-treated astrocytes activated naive, transgenic OVA-specific T cells. Astrocyte-induced activation of both OVA-specific naive T cells and activated Th1 cells was dependent primarily on B7-2-mediated costimulation, as proliferation was inhibited by CTLA4-Ig and by anti-B7-2 mAbs. These results suggest that astrocytes in an inflammatory environment have the capacity to express the required MHC class II and B7 costimulatory molecules necessary for efficient activation of naive T cells. Since we have shown that T cells specific for endogenous myelin epitopes released during acute EAE play the major pathologic effector role in subsequent disease relapses (epitope spreading), astrocytes could play a role in the local activation and expansion of these responses.

T-cell responses to enteric antigens.

Antigens that enter through the intestine induce both local and systemic immune responses by mechanisms that are not well understood. Recent work has established the lamina propria as a immunologically potent compartment, capable of stimulating T cells to antigens delivered from the lumen into the tissue. Several of these studies have characterized the immune potential of lamina propria antigen presenting cell populations, such as macrophages and dendritic cells, as well as the T-cell responses to lumenal infections and parasites. To study the responses of distinct populations of CD4+ T cells to enteric antigens, we have used a T-cell receptor (TCR) transgenic system. First, these studies have shown that intestinal lamina propria T cells represent a unique population of previously activated cells that have been primed in vivo to deliver a wide range of effector functions compared with T cells in the lymphoid tissue. Second, by following activated transgenic T cells with anti-TCR monoclonal antibody, we observed that activation of antigen-specific T cells in the periphery results in the trafficking of distinct subsets to mucosal Peyer's patch and lamina propria compartments. The mechanisms that may explain this trafficking phenomenon are only now becoming clear, with recent work from several laboratories suggesting that a multistep hypothesis, involving the function of various surface selectin and integrin molecules, plays an important role. With these current results, we hypothesize that mucosal and systemic immune responses to oral proteins are determined by the activational state and surface marker phenotype of T-cell subsets that respond. We speculate that a better understanding of these T-cell responses could help develop effective oral vaccination regimens.

Differential recognition of peptide analogs by naive verses activated PLP 139-151-specific CD4+ T cells.

CD4+ T cells specific for PLP 139-151 induce a relapsing-remitting form of EAE which is similar to the human demyelinating disease multiple sclerosis (MS) in both clinical course and histopathology. Conservative and nonconservative amino acid substitutions were introduced at three TcR or MHC contact residues within PLP 139-151 to identify fine specificity requirements, at the polyclonal level, for stimulating naive encephalitogenic T cells and for reactivating pre-primed autoreactive T cells as measured by T cell proliferation, cytokine induction, and functional encephalitogenic potential. The results indicate that peptides with substitutions at position 145 exhibited a significantly diminished ability to induce active disease, but these substitutions had little or no effect on the ability to activate PLP 139-151-primed T cells for proliferation or disease transfer. A conservative or a nonconservative substitution at position 144 ablated both encephalitogenic potential in active and adoptive EAE models and the ability to induce proliferative responses in T cells primed to the native peptide. A nonconservative lysine for glycine, but not a conservative serine substitution, at position 146 had similar effects. In contrast to their inability to induce active EAE and stimulate in vitro proliferation of PLP 139-151-primed T cells, the Y144 and the 146 analog peptides were able to suboptimally reactivate these cells for transfer of adoptive EAE. Furthermore, the nonencephalitogenic K146 peptide was found to exacerbate in vivo induction of EAE induced by priming with a suboptimal dose of PLP 139-151. These data support the hypothesis that naive neuroantigen-specific CD4+ T cells have more stringent activation requirements than do PLP 139-151-specific T cells which have previously encountered antigen. The finding that the analog peptides induced differential patterns of cytokine production, with LT/TNF-alpha production but not IFN-gamma production correlating with full encephalitogenic potential, suggests different functional outcomes may result from differential levels of signal transduction triggered by the substituted peptides. The significance of these results to the potential development of autoimmune disease via molecular mimicry and for the development of new strategies for preventing and treating T cell-mediated autoimmune diseases is discussed.