In this investigation, we have examined the integrated relationship between IL-13, IL-4, and IL-5 for the development of airways hyperreactivity (AHR) in a model of asthma in BALB/c mice. Sensitization and aeroallergen challenge of both wild-type (WT) and IL-13 gene-targeted (IL-13−/−) mice induced allergic disease that was characterized by pulmonary eosinophilia and AHR to β-methacholine. Although these responses in IL-13−/− mice were heightened compared with WT, they could be reduced to the level in nonallergic mice by the concomitant neutralization of IL-4. Mice in which both IL-4 and IL-13 were depleted displayed a marked reduction in tissue eosinophils, despite the development of a blood eosinophilia. Similar neutralization of IL-4 in WT mice only partially reduced AHR with no effect on tissue eosinophilia. In addition, neutralization of IL-5 in IL-13−/− mice, but not in WT mice, inhibited AHR, suggesting that tissue eosinophilia is linked to the mechanism underlying AHR only in the absence of IL-13. Additionally, mucus hypersecretion was attenuated in IL-13−/− mice, despite the persistence of AHR. Taken together, our data suggest both a modulatory role for IL-13 during sensitization and a proinflammatory role during aeroallergen challenge. The latter process appears redundant with respect to IL-4.

Interleukin (IL)-5 and IL-13 are thought to play key roles in the pathogenesis of asthma. Although both cytokines use eotaxin to regulate eosinophilia, IL-13 is thought to operate a separate pathway to IL-5 to induce airways hyperreactivity (AHR) in the allergic lung. However, identification of the key pathway(s) used by IL-5 and IL-13 in the disease process is confounded by the failure of anti–IL-5 or anti–IL-13 treatments to completely inhibit the accumulation of eosinophils in lung tissue. By using mice deficient in both IL-5 and eotaxin (IL-5/eotaxin−/−) we have abolished tissue eosinophilia and the induction of AHR in the allergic lung. Notably, in mice deficient in IL-5/eotaxin the ability of CD4+ T helper cell (Th)2 lymphocytes to produce IL-13, a critical regulator of airways smooth muscle constriction and obstruction, was significantly impaired. Moreover, the transfer of eosinophils to IL-5/eotaxin−/− mice overcame the intrinsic defect in T cell IL-13 production. Thus, factors produced by eosinophils may either directly or indirectly modulate the production of IL-13 during Th2 cell development. Our data show that IL-5 and eotaxin intrinsically modulate IL-13 production from Th2 cells and that these signaling systems are not necessarily independent effector pathways and may also be integrated to regulate aspects of allergic disease.

Asthma pathophysiology is intimately regulated by CD4؉ Th2 lymphocytes and the cytokines interleukin (IL)-4 and IL-13. However, the mechanisms by which these cytokines promote disease have not been fully elucidated. In order to identify novel molecular mediators of allergy, a comparison was made of the bronchoalveolar lavage, which demonstrated that the Ym2 protein was abundantly up-regulated in the lung during the development of allergy. Low levels of the Ym1 isomer were also detected. Importantly, neither Ym1 nor Ym2 has been characterized previously in the context of allergic pulmonary inflammation. Western immunoblot showed that enhanced expression of these proteins was dependent on CD4؉ T cells and IL-4 or IL-13 signaling via the IL-4R␣ subunit. In addition, intratracheal instillation of IL-13 into naive mice was sufficient to induce expression. Ym1 is homologous to eosinophil chemotactic factor L. However, only weak eosinophil chemotaxis was observed in response to Ym protein in both in vitro and in vivo assays. By contrast, the homology of Ym1 and Ym2 to proteins associated with tissue remodeling, together with the previous findings that Ym1 is homologous to chitinase and binds heparin sulfate and GlcN oligomers (chitobiose, chitotriose, and chitotetraose), strongly suggests these proteins play an important role in airway wall remodeling in the allergic lung.

In this review we identify the elemental signals that regulate eosinophil accumulation in the allergic lung. We show that there are two interwoven mechanisms for the accumulation of eosinophils in pulmonary tissues and that these mechanisms are linked to the development of airways hyperreactivity (AHR). Interleukin-(IL)-5 plays a critical role in the expansion of eosinophil pools in both the bone marrow and blood in response to allergen provocation of the airways. Secondly, IL-4 and IL-13 operate within the allergic lung to control the transmigration of eosinophils across the vascular bed into pulmonary tissues. This process exclusively promotes tissue accumulation of eosinophils. IL-13 and IL-4 probably act by activating eosinophil-specific adhesion pathways and by regulating the production of IL-5 and eotaxin in the lung compartment. IL-5 and eotaxin co-operate locally in pulmonary tissues to selectively and synergistically promote eosinophilia. Thus, IL-5 acts systemically to induce eosinophilia and within tissues to promote local chemotactic signals. Regulation of IL-5 and eotaxin levels within the lung by IL-4 and IL-13 allows Th2 cells to elegantly co-ordinate tissue and peripheral eosinophilia. Whilst the inhibition of either the IL-4/IL-13 or IL-5/eotaxin pathways resulted in the abolition of tissue eosinophils and AHR, only depletion of IL-5 and eotaxin concurrently results in marked attenuation of pulmonary inflammation. These data highlight the importance of targeting both IL-5 and CCR3 signalling systems for the resolution of inflammation and AHR associated with asthma.

Escherichia coli contains two major systems for transporting inorganic phosphate (P i ). The low-affinity P i transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the highaffinity Pst system (pstSCAB) is induced at low external P i concentrations by the pho regulon and is an ABC transporter. We isolated a third putative P i transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional P i transporter that may be repressed at low P i levels by the pho regulon. While a pitB ؉ cosmid clone allowed growth on medium containing 500 M P i , E. coli with wild-type genomic pitB (pitA ⌬pstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB ⌬pstC345 triple mutant. The mutation ⌬pstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB ؉ pitA ⌬pstC345 strain to utilize P i , with P i uptake rates significantly higher than background levels. In addition, the apparent K m of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.Escherichia coli contains at least two major systems for transporting inorganic phosphate (P i ). The low-affinity inorganic phosphate transporter (Pit) is dependent on the proton motive force for energy and is constitutively expressed (30,31,49). When P i is plentiful, this is the major uptake system for phosphate, with a reported apparent K m (K m app ) of 25 M (30) to 38 M (50) in whole cells and 11.9 M in membrane vesicles (43). If the external P i concentration is below the millimolar range, the high-affinity phosphate-specific transport (Pst) system is induced. This has a K m app of around 0.2 M (30, 50). The Pst system is a complex of four proteins, including a periplasmic binding protein, which is energized by ATP and belongs to the ABC transporter family (7,15,47). The pst operon contains five genes under pho regulon control (1,40,41), which induces a range of genes when the phosphate supply is limited. Both the Pit and Pst systems are highly specific for P i (30). Another two transporters accept P i as a low-affinity analogue for either glycerol-3-phosphate (glpT) (18) or glucose-6-phosphate (uhpT) (29, 53), but in the absence of Pit and Pst activity, these latter two systems cannot support cell growth when supplied with P i (38).Divalent cations, such as Mg 2ϩ or Ca 2ϩ