A protective role for periostin and TGF-ß in IgE-mediated allergy and airway hyperresponsiveness.

BACKGROUND: The pathophysiology of asthma involves allergic inflammation and remodelling in the airway and airway hyperresponsiveness (AHR) to cholinergic stimuli, but many details of the specific underlying cellular and molecular mechanisms remain unknown. Periostin is a matricellular protein with roles in tissue repair following injury in both the skin and heart. It has recently been shown to be up-regulated in the airway epithelium of asthmatics and to increase active TGF-ß. Though one might expect periostin to play a deleterious role in asthma pathogenesis, to date its biological role in the airway is unknown. OBJECTIVE: To determine the effect of periostin deficiency on airway responses to inhaled allergen. METHODS: In vivo measures of airway responsiveness, inflammation, and remodelling were made in periostin deficient mice and wild-type controls following repeated intranasal challenge with Aspergillus fumigatus antigen. In vitro studies of the effects of epithelial cell-derived periostin on murine T cells were also performed. RESULTS: Surprisingly, compared with wild-type controls, periostin deficient mice developed increased AHR and serum IgE levels following allergen challenge without differences in two outcomes of airway remodelling (mucus metaplasia and peribronchial fibrosis). These changes were associated with decreased expression of TGF-ß1 and Foxp3 in the lungs of periostin deficient mice. Airway epithelial cell-derived periostin-induced conversion of CD4(+) CD25(-) cells into CD25(+) , Foxp3(+) T cells in vitro in a TGF-ß dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Allergen-induced increases in serum IgE and bronchial hyperresponsiveness are exaggerated in periostin deficient mice challenged with inhaled aeroallergen. The mechanism of periostin's effect as a brake on allergen-induced responses may involve augmentation of TGF-ß-induced T regulatory cell differentiation.

Accumulation of eosinophils in intestine-draining mesenteric lymph nodes occurs after Trichuris muris infection.

Eosinophils have recently been demonstrated capable of localizing to lymph nodes that drain mucosal surfaces, in particular during T helper 2 (Th2) responses. Resistance of mice to infection with the gastrointestinal nematode Trichuris muris depends critically on mounting of a Th2 response and represents a useful model system to investigate Th2 responses. Following infection of resistant BALB/c mice with T. muris, we observed accumulation of eosinophils in intestine-draining mesenteric lymph nodes (MLNs). The accumulation of MLN eosinophils was initiated during the second week of infection and peaked during worm expulsion. In contrast, we detected a comparably late and modest increase in eosinophil numbers in the MLNs of infected susceptible AKR mice. MLN eosinophils localized preferentially to the medullary region of the lymph node, displayed an activated phenotype and contributed to the interleukin-4 (IL-4) response in the MLN. Despite this, mice genetically deficient in eosinophils efficiently generated IL-4-expressing CD4(+) T cells, produced Th2 cytokines and mediated worm expulsion during primary T. muris infection. Thus, IL-4-expressing eosinophils accumulate in MLNs of T. muris-infected BALB/c mice but are dispensable for worm expulsion and generation of Th2 responses, suggesting a distinct or subtle role of MLN eosinophils in the immune response to T. muris infection.

Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection.

Leishmania major are intramacrophage parasites whose eradication requires the induction of T helper 1 (Th1) effector cells capable of activating macrophages to a microbicidal state. Interleukin 12 (IL-12) has been recently identified as a macrophage-derived cytokine capable of mediating Th1 effector cell development, and of markedly enhancing interferon gamma (IFN-gamma) production by T cells and natural killer cells. Infection of macrophages in vitro by promastigotes of L. major caused no induction of IL-12 p40 transcripts, whereas stimulation using heat-killed Listeria or bacterial lipopolysaccharide induced readily detectable IL-12 mRNA. Using a competitor construct to quantitate a number of transcripts, a kinetic analysis of cytokine induction during the first few days of infection by L. major was performed. All strains of mice examined, including susceptible BALB/c and resistant C57BL/6, B10.D2, and C3H/HeN, had the appearance of a CD4+ population in the draining lymph nodes that contained transcripts for IL-2, IL-4, and IFN-gamma (and in some cases, IL-10) that peaked 4 d after infection. In resistant mice, the transcripts for IL-2, IL-4, and IL-10 were subsequently downregulated, whereas in susceptible BALB/c mice, these transcripts were only slightly decreased, and IL-4 continued to be reexpressed at high levels. IL-12 transcripts were first detected in vivo by 7 d after infection, consistent with induction by intracellular amastigotes. Challenge of macrophages in vitro confirmed that amastigotes, in contrast to promastigotes, induced IL-12 p40 mRNA. Reexamination of the cytokine mRNA at 4 d revealed expression of IL-13 in all strains analyzed, suggesting that IL-2 and IL-13 may mediate the IL-12-independent production of IFN-gamma during the first days after infection. Leishmania have evolved to avoid inducing IL-12 from host macrophages during transmission from the insect vector, and cause a striking induction of mRNAs for IL-2, IL-4, IL-10, and IL-13 in CD4+ T cells. Each of these activities may favor survival of the organism.

CD4+ effector cells default to the Th2 pathway in interferon gamma-deficient mice infected with Leishmania major.

Mice with homologous disruption of the interferon gamma (IFN-gamma) gene on the C57BL/6 background were infected with Leishmania major and the immune response assessed. In contrast to wild-type or heterozygous knockout mice, deficient animals were unable to restrict growth of the parasite and suffered lethal infection over 6-8 wk. Although wild-type and heterozygous littermates developed CD4+ cells that contained transcripts for IFN-gamma and lymphotoxin, typical of T helper type 1 (Th1) cells, the knockout mice developed CD4+ cells that contained transcripts for interleukin 4 (IL-4), IL-5, and IL-13, typical of Th2 cells. ELISPOT assays confirmed the reciprocal patterns of IFN-gamma or IL-4 production by T cells in similar frequencies in the respective groups of mice, and antibody analysis confirmed the presence of Th2-mediated isotype switching in the knockout mice. These data suggest that CD4+ T cells that normally respond to antigens by differentiation to Th1 cells default to the Th2 pathway in the absence of endogenous IFN-gamma.

Genetic regulation of commitment to interleukin 4 production by a CD4(+) T cell-intrinsic mechanism.

The dysregulated expression of interleukin 4 (IL-4) can have deleterious effects on the outcome of infectious and allergic diseases. Despite this, the mechanisms by which naive T cells commit to IL-4 expression during differentiation into mature effector cells remain incompletely defined. As compared to cells from most strains of mice, activated CD4(+) T cells from BALB mice show a bias towards IL-4 production and T helper 2 commitment in vitro and in vivo. Here, we show that this bias arises not from an increase in the amount of IL-4 produced per cell, but rather from an increase in the proportion of CD4(+) T cells that commit to IL-4 expression. This strain-specific difference in commitment was independent of signals mediated via the IL-4 receptor and hence occurred upstream of potential autoregulatory effects of IL-4. Segregation analysis of the phenotype in an experimental backcross cohort implicated a polymorphic locus on chromosome 16. Consistent with a role in differentiation, expression of the phenotype was CD4(+) T cell intrinsic and was evident as early as 16 h after the activation of naive T cells. Probabilistic gene activation is proposed as a T cell-intrinsic mechanism capable of modulating the proportion of naive T cells that commit to IL-4 production.

Interferon gamma derived from CD4(+) T cells is sufficient to mediate T helper cell type 1 development.

Interferon gamma (IFN-gamma) has been implicated in T helper type 1 (Th1) cell development through its ability to optimize interleukin 12 (IL-12) production from macrophages and IL-12 receptor expression on activated T cells. Various systems have suggested a role for IFN-gamma derived from the innate immune system, particularly natural killer (NK) cells, in mediating Th1 differentiation in vivo. We tested this requirement by reconstituting T cell and IFN-gamma doubly deficient mice with wild-type CD4(+) T cells and challenging the mice with pathogens that elicited either minimal or robust IL-12 in vivo (Leishmania major or Listeria monocytogenes, respectively). Th1 cells developed under both conditions, and this was unaffected by the presence or absence of IFN-gamma in non-T cells. Reconstitution with IFN-gamma-deficient CD4(+) T cells could not reestablish control over L. major, even in the presence of IFN-gamma from the NK compartment. These data demonstrate that activated T cells can maintain responsiveness to IL-12 through elaboration of endogenous IFN-gamma without requirement for an exogenous source of this cytokine.

Myristyl acylation of the tumor necrosis factor alpha precursor on specific lysine residues.

NH2-terminal glycine myristyl acylation is a cotranslational modification that affects both protein localization and function. However, several proteins that lack NH2-terminal glycine residues, including the interleukin 1 (IL-1) precursors, also contain covalently linked myristate. To date, the site(s) of acylation of these proteins has not been determined. During an evaluation of IL-1 acylation, it was observed that [3H]myristate-labeled human monocyte lysates contained a prominent 26-kD myristylated protein, which was identified as the tumor necrosis factor alpha (TNF) precursor protein on the basis of specific immune precipitation. Radioimmunoprecipitates from the supernates of labeled monocytes indicated that the processed or mature 17-kD form of TNF does not contain myristate, suggesting that the site of acylation occurs within the 76-amino acid propiece of the precursor molecule. As the TNF precursor does not contain an NH2-terminal glycine, we hypothesized that myristyl acylation occurs on the N-epsilon-NH2 groups of lysine, of which two are present in the propiece (K19K20). Synthetic peptides were designed to include all seven lysine residues present within the entire 26-kD TNF precursor, and used in an in vitro myristyl acylation assay containing peptide, myristyl-CoA, and monocyte lysate as a source of enzyme. Analysis of reaction products by reverse phase high performance liquid chromatography and gas phase sequencing demonstrated the exclusive myristyl acylation of K19 and K20, consistent with the presence in monocytes of a specific lysyl N-epsilon-NH2-myristyl transferase activity. The acylated lysine residues are located immediately downstream from a hydrophobic, probable membrane-spanning segment of the propiece. Specific myristyl acylation of the TNF propiece may facilitate membrane insertion or anchoring of this critical inflammatory mediator.

Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets.

We purified poly(A)+ mRNA from the spleen and lymph nodes at designated times after infection with Leishmania major in genetically susceptible BALB/c and resistant C57BL/6 mice. The steady-state levels of IL-2, IFN-gamma, IL-4, and IL-1 beta mRNA were determined using Northern hybridizations. IL-2 mRNA levels in the infected organs of BALB/c and C57BL/6 mice were comparable after infection, but IFN-gamma and IL-4 mRNA levels were reciprocally expressed. Levels of IFN-gamma mRNA in C57BL/6 draining nodes and spleen were significantly greater than in BALB/c mice except at 4 and 6 wk of infection, when splenic IFN-gamma mRNA levels were transiently comparable. In contrast, IL-4 mRNA was apparent only in BALB/c and not in C57BL/6 nodes and spleen. Tissue levels of IL-1 beta mRNA were 10-20-fold greater in BALB/c mice. BALB/c mice were pretreated with GK1.5 mAb, a manipulation that promotes healing of subsequent infection by transiently depleting L3T4+ cells. At 8 wk of infection, by which time lymphoid organs were repopulated with L3T4+ cells, GK1.5-pretreated BALB/c mice produced IFN-gamma, but not IL-4 message. Serum levels of IgE were markedly elevated in infected BALB/c, but not in infected C57BL/6 or GK1.5-pretreated BALB/c mice, consistent with in vivo biologic activity of IL-4 in nonhealing mice. Treatment of infected BALB/c mice with neutralizing anti-IL-4 antibody abolished the elevation of serum IgE and significantly attenuated the progression of disease as assessed by size and ulceration of the lesion, and by reduction in the number of tissue parasites. Both protective and deleterious responses to Leishmania infection have previously been shown to be L3T4+ cell dependent. Our findings are consistent with the differential expansion of protective, IFN-gamma-producing Th1 cells in healing mice, and the expansion of deleterious, IL-4-producing Th2 cells in nonhealing mice. The inverse relationship of IFN-gamma and IL-4 gene expression during leishmaniasis may underlie the divergence of cellular and humoral immunity that occurs during chronic infection with Leishmania and possibly other intracellular parasites.

Human placental cytotrophoblasts produce the immunosuppressive cytokine interleukin 10.

The mechanism by which the mammalian mother accepts the implanting fetus as an allograft remains unexplained, but is likely to be the result of a combination of factors. Mononuclear cytotrophoblasts, the specialized fetal cells of the placenta that invade the uterus, play an important role. These cells express HLA-G, an unusual major histocompatibility complex class I-B molecule, and secrete cytokines and pregnancy-specific proteins that can regulate immune function. We investigated whether cytotrophoblasts secrete interleukin 10 (IL-10), a cytokine that potently inhibits alloresponses in mixed lymphocyte reactions. Cytotrophoblasts from all stages of pregnancy produced IL-10 in vitro, but neither placental fibroblasts nor choriocarcinoma (malignant trophoblast) cell lines did so. Spontaneous IL-10 production averaged 650, 853, and 992 pg/10(6) cells in the first, second, and third trimesters of pregnancy, respectively. IL-10 secretion dropped approximately 10-fold after the first 24 h of culture, and was paralleled by a decrease in messenger RNA. IL-10 messenger RNA was detected in biopsies of the placenta and the portion of the uterus that contains invasive cytotrophoblasts, suggesting that this cytokine is also produced in vivo. IL-10 secreted by cytotrophoblasts in vitro is bioactive, as determined by its ability to suppress interferon gamma production in an allogeneic mixed lymphocyte reaction. We conclude that human cytotrophoblast IL-10 may be an important factor that contributes to maternal tolerance of the allogeneic fetus.

T helper subset differentiation in the absence of invariant chain.

The outcome of murine infection with Leishmania major is regulated by major histocompatibility complex class II-restricted T helper cells. Invariant chain-deficient (Ii -/-) mice have impaired ability to present major histocompatibility complex class II-restricted antigens, and reduced numbers of CD4+ T cells. Despite these deficits, C57BL/6 Ii -/- mice controlled L. major infection comparably to wild-type mice. As assessed by mRNA analysis and in vitro antigen restimulation for IFN-gamma, Ii -/- mice had normal induction of Th1 subset differentiation even though antigen-dependent proliferation of their lymph node cells was substantially compromised. In addition, BALB/c Ii -/- mice exhibited a progressive course of infection and Th2 effector cell development that were comparable to that seen in wild-type BALB/c mice. We wished to determine whether this unexpected efficiency of T helper subset induction despite inefficient T cell stimulation could be modeled in vitro. In the presence of rIL-12 or rIL-4 naive parasite-specific transgenic T cells could mature into IFN-gamma-or IL-4-secreting T helper cells, respectively, even when antigen presentation was suboptimal or antigen dose was submitogenic. These experiments demonstrate that activation of T helper cells to a threshold required for IL-2 production or proliferation is not required to achieve induction of disease-regulating T helper cell effector functions, and that pathogen-associated secondary activation signals may facilitate the full differentiation of T helper subsets during limiting presentation of antigenic peptides.

Cure of murine leishmaniasis with anti-interleukin 4 monoclonal antibody. Evidence for a T cell-dependent, interferon gamma-independent mechanism.

BALB/c mice infected with Leishmania major develop fatal, progressive disease, despite an immune response characterized by expansion of CD4+ T cells in the draining lymph nodes. The immune response has been further characterized by a lack of IFN-gamma mRNA, but increased IL-4 mRNA in lymphoid tissues, and striking elevation of serum IgE. Treatment of infected BALB/c mice with rIFN-gamma at doses shown to be beneficial in other protozoan infections was insufficient to ameliorate L. major infection. In contrast, neutralization of IL-4 by six weekly injections of mAb 11B11 led to attenuation of disease in 100% of animals, and complete cure in 85%. Resolution of disease required the presence of T cells, and recovered mice remained resistant to reinfection at 12 wk. This immunity was adoptively transferable and was dependent on both CD4+ and CD8+ cells. Although administration of anti-IL-4 was associated with fourfold increase in IFN-gamma mRNA in lymph node cells draining the lesion, the coadministration of neutralizing R4 6A2 anti-IFN-gamma mAb had no effect on resistance to disease. This was in marked contrast to resolution of disease in both resistant C57BL/6- and GK1.5-pretreated BALB/c mice that was abrogated by in vivo treatment with anti-IFN-gamma. These data suggest a novel mechanism of cellular immunity established by interference with the development of Th2 cells during infection.

Interleukin 4, but not interleukin 5 or eosinophils, is required in a murine model of acute airway hyperreactivity.

Reversible airway hyperreactivity underlies the pathophysiology of asthma, yet the precise mediators of the response remain unclear. Human studies have correlated aberrant activation of T helper (Th) 2-like effector systems in the airways with disease. A murine model of airway hyperreactivity in response to acetylcholine was established using mice immunized with ovalbumin and challenged with aerosolized antigen. No airway hyperractivity occurred in severe combined immunodeficient mice. Identically immunized BALB/c mice developed an influx of cells, with a predominance of eosinophils and CD4+ T cells, into the lungs and bronchoalveolar lavage fluid at the time that substantial changes in airway pressure and resistance were quantitated. Challenged animals developed marked increases in Th2 cytokine production, eosinophil influx, and serum immunoglobulin E levels. Neutralization of interleukin (IL) 4 using monoclonal antibodies administered during the period of systemic immunization abrogated airway hyperractivity but had little effect on the influx of eosinophils. Administration of anti-IL-4 only during the period of the aerosol challenge did not affect the subsequent response to acetylcholine. Finally, administration of anti-IL-5 antibodies at levels that suppressed eosinophils to < 1% of recruited cells had no effect on the subsequent airway responses. BALB/c mice had significantly greater airway responses than C57BL/6 mice, consistent with enhanced IL-4 responses to antigen in BALB/c mice. Taken together, these data implicate IL-4 generated during the period of lymphocyte priming with antigen in establishing the cascade of responses required to generate airway hyperractivity to inhaled antigen. No role for IL-5 or eosinophils could be demonstrated.

Altered immune responses in interleukin 10 transgenic mice.

Interleukin (IL)-10 is a pleiotropic cytokine which inhibits a broad array of immune parameters including T helper cell type 1 (Th1) cytokine production, antigen presentation, and antigen-specific T cell proliferation. To understand the consequences of altered expression of IL-10 in immune models of autoimmune disease, the response to infectious agents, and the response to tumors, we developed transgenic mice expressing IL-10 under the control of the IL-2 promoter. Upon in vitro stimulation, spleen cells from unimmunized transgenic mice secrete higher levels of IL-10 and lower amounts of IFN-gamma than do controls, although no gross abnormalities were detected in lymphocyte populations or serum Ig levels. Transfer of normally pathogenic CD4(+) CD45RBhigh splenic T cells from IL-10 transgenic mice did not cause colitis in recipient severe combined immunodeficiency mice. Furthermore, co-transfer of these transgenic cells with CD4(+) CD45RBhigh T cells from control mice prevented disease. Transgenic mice retained their resistance to Leishmania major infection, indicating that their cell-mediated immune responses were not globally suppressed. Lastly, in comparison to controls, IL-10 transgenic mice were unable to limit the growth of immunogenic tumors. Administration of blocking IL-10 mAbs restored in vivo antitumor responses in the transgenic mice. These results demonstrate that a single alteration in the T cell cytokine profile can lead to dramatic changes in immune responses in a manner that is stimulus dependent. These mice will be useful in defining differences in inflammatory conditions and cellular immunity mediated by IL-10.

Beta 2-microglobulin-dependent NK1.1+ T cells are not essential for T helper cell 2 immune responses.

A number of investigations have established the critical role of interleukin 4 (IL-4) in mediating the development of T helper (Th)2 effector cells in vitro and in vivo. Despite intensive study, the origin of the IL-4 required for Th2 priming and differentiation remains unclear. Natural killer (NK)1.1+ alpha/beta T cell receptor+ T(NT) cells, a unique lineage of cells capable of producing large amounts of IL-4 after activation in vivo, are important candidates for directing Th2 priming. These cells are selected by the nonpolymorphic major histocompatibility complex (MHC) class I molecule, CD1, and are deficient in beta 2-microglobulin (beta 2m)-null mice. We used beta 2m-deficient mice on both BALB/c and C57BL/6 backgrounds to examine their capacity to mount Th2 immune responses after challenge with a number of well-characterized antigens administered by a variety of routes. As assessed by immunization with protein antigen, infection with Leishmania major, embolization with eggs of Schistosoma mansoni, intestinal infection with Nippostrongylus brasiliensis, or induction of airway hyperreactivity to aerosolized antigen, beta 2m-deficient mice developed functional type 2 immune responses that were not substantially different than those in wild-type mice. Production of IL-4 and the generation of immunoglobulin E (IgE) and eosinophil responses were preserved as assessed by a variety of assays. Collectively, these results present a comprehensive analysis of type 2 immune responses in beta 2m-deficient mice, and indicate that beta 2m-dependent NT cells are not required for Th2 development in vivo.

Altered ligands reveal limited plasticity in the T cell response to a pathogenic epitope.

Experimental leishmaniasis offers a well characterized model of T helper type 1 cell (Th1)-mediated control of infection by an intracellular organism. Susceptible BALB/c mice aberrantly develop Th2 cells in response to infection and are unable to control parasite dissemination. The early CD4(+) T cell response in these mice is oligoclonal and reflects the expansion of Vbeta4/ Valpha8-bearing T cells in response to a single epitope from the parasite Leishmania homologue of mammalian RACK1 (LACK) antigen. Interleukin 4 (IL-4) generated by these cells is believed to direct the subsequent Th2 response. We used T cells from T cell receptor-transgenic mice expressing such a Vbeta4/Valpha8 receptor to characterize altered peptide ligands with similar affinity for I-Ad. Such altered ligands failed to activate IL-4 production from transgenic LACK-specific T cells or following injection into BALB/c mice. Pretreatment of susceptible mice with altered peptide ligands substantially altered the course of subsequent infection. The ability to confer a healer phenotype on otherwise susceptible mice using altered peptides that differed by a single amino acid suggests limited diversity in the endogenous T cell repertoire recognizing this antigen.

Independent and epigenetic regulation of the interleukin-4 alleles in CD4+ T cells.

How an individual effector T cell acquires a particular cytokine expression pattern from many possible patterns remains unclear. CD4+ T cells from F1 mice, which allowed assignment of the parental origin of interleukin-4 (IL-4) transcripts, were divided into clones that expressed IL-4 biallelically or monoallelically from either allele. The allelic pattern was transmitted as a stable epigenetic trait. Regulation of cytokine expression by a mechanism that treats each allele independently suggests a probabilistic process by which a diverse repertoire of combinatorially assorted cytokine gene expression patterns could be generated among the clonally related daughters of a single precursor cell.

The instructive role of innate immunity in the acquired immune response.

Innate immunity has been considered only to provide rapid, incomplete antimicrobial host defense until the slower, more definitive acquired immune response develops. However, innate immunity may have an additional role in determining which antigens the acquired immune system responds to and the nature of that response. Knowledge of the molecules and pathways involved may create new therapeutic options for infectious and autoimmune diseases.

TH1 and TH2 cell antigen receptors in experimental leishmaniasis.

The complexity and chronicity of parasitic infections have obscured the identification of biologically relevant antigens. Analysis of the T cell receptor repertoire used by mice infected with Leishmania major revealed the expansion of a restricted population of CD4+ cells. These cells expressed the V alpha 8-J alpha TA72, V beta 4 heterodimer in both progressive infection and protective immunity and across several major histocompatibility haplotypes. Thus, the same immunodominant parasite epitope drives the disparate outcomes of this infectious process, suggesting that candidate vaccine antigens selected by screening of immune individuals may be capable of exacerbating disease in genetically susceptible individuals.

Helper T cells without CD4: control of leishmaniasis in CD4-deficient mice.

Expression of either the CD4 or CD8 glycoproteins discriminates two functionally distinct lineages of T lymphocytes. A null mutation in the gene encoding CD4 impairs the development of the helper cell lineage that is normally defined by CD4 expression. Infection of CD4-null mice with Leishmania has revealed a population of functional helper T cells that develops despite the absence of CD4. These CD8- alpha beta T cell receptor+ T cells are major histocompatibility complex class II-restricted and produce interferon-gamma when challenged with parasite antigens. These results indicate that T lymphocyte lineage commitment and peripheral function need not depend on the function of CD4.

Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons.

Long-range regulatory elements are difficult to discover experimentally; however, they tend to be conserved among mammals, suggesting that cross-species sequence comparisons should identify them. To search for regulatory sequences, we examined about 1 megabase of orthologous human and mouse sequences for conserved noncoding elements with greater than or equal to 70% identity over at least 100 base pairs. Ninety noncoding sequences meeting these criteria were discovered, and the analysis of 15 of these elements found that about 70% were conserved across mammals. Characterization of the largest element in yeast artificial chromosome transgenic mice revealed it to be a coordinate regulator of three genes, interleukin-4, interleukin-13, and interleukin-5, spread over 120 kilobases.