Lymphocyte-specific reconstitution of IL-4Ra-deficient mice : characterization and infectious disease studies (original) (raw)
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Proceedings of the National Academy of Sciences, 1997
IL-4 receptor ␣ chain (IL-4R␣)-deficient mice were generated by gene-targeting in BALB͞c embryonic stem cells. Mutant mice showed a loss of IL-4 signal transduction and functional activity. The lack of IL-4R␣ resulted in markedly diminished, but not absent, TH2 responses after infection with the helminthic parasite Nippostrongylus brasiliensis. CD4 ؉ , CD62L-high, and CD62L-low T cell populations from uninfected IL-4R␣ ؊/؊ mice were isolated by cell sorting. Upon primary stimulation by T cell receptor cross-linkage, the CD62L-low, but not the CD62L-high, cells secreted considerable amounts of IL-4, which was strikingly enhanced upon 4-day culture with anti-CD3 in the presence or absence of IL-4. CD62L-low cells isolated from IL-4R␣ ؊/؊ ,  2-microglobulin ؊/؊ double homozygous mice produced less IL-4 than did either IL-4R␣ ؊/؊ or wild-type mice. These results indicate that an IL-4-independent,  2-microglobulin-dependent pathway exists through which the CD62L-low CD4 ؉ population has acquired IL-4-producing capacity in vivo, strongly suggesting that these cells are NK T cells.
Regulation of the Expression of the Soluble and Membrane Forms of the Murine IL-4 Receptor
Cellular Immunology, 1997
switch to the IgG1 and IgE isotypes; the differentiation of precursor T helper cells into mature Th2 cells; and The actions of interleukin-4 (IL-4) in vivo are likely inhibitory effects on the production of proinflammatory to be positively influenced by the expression of memcytokines by monocytes (3-8). High-affinity receptors brane IL-4 receptors (mIL-4R) on target cells and negatively by the concentration of soluble IL-4 receptors for IL-4 (IL-4R) are expressed on a wide variety of he-(sIL-4R) in the extracellular environment. Inasmuch mopoietic and nonhemopoietic cells and are composed as the two forms of the mouse IL-4R are differentially of a 140-kDa ligand-binding chain (IL-4Ra) in associaencoded by alternatively spliced mRNA transcripts, tion with the common g chain, a signal transducing the purpose of this work was to determine how their subunit shared by several cytokine receptor complexes expression is regulated by IL-4 and T cell activation including IL-2R, IL-7R, IL-9R, and IL-15R (9-14). In and whether there is preferential expression of one addition, a second class of IL-4R has been reported to type of transcript over the other. In this study, the be formed by the interaction of the IL-4Ra chain with expression of sIL-4R and mIL-4R transcripts was anathe IL-13Ra chain (15, 16). lyzed by a semiquantitative RT-PCR method in resting The IL-4Ra chain is produced naturally both as a and mitogen-activated splenic cells. Irrespectively of membrane-associated and as a truncated, soluble form the state of cell activation, IL-4 up-regulated the levels (mIL-4R and sIL-4R, respectively) (17-19). The two ILof both types of mRNA with similar kinetics and dose-4R forms are encoded by distinct mRNA species that response curves. In contrast, ConA failed to enhance arise from alternative splicing of a primary transcript the steady-state levels of sIL-4R or mIL-4R transcripts despite increased expression at the protein level, sug-of the IL-4R gene (7). The difference between the two gesting that sIL-4R expression is also regulated at lev-mRNAs is the splicing out of sequences corresponding els other than transcription. Western blot analysis of to exon 8 in the membrane IL-4R-specific mRNA (20). supernatants of IL-4-and ConA-stimulated spleen cells Retention of exon 8 in the sIL-4R-specific transcript substantiated the presence of sIL-4R molecules deresults in the addition of six novel amino acids followed rived by translation of sIL-4R-specific transcripts, thus by premature translational termination immediately confirming the importance of this mechanism for the upstream of the transmembrane domain region generation of sIL-4R molecules in normal cells. These (17, 20). The sIL-4R protein, therefore, consists of the results indicate that the sIL-4R-and mIL-4R-specific extracytoplasmic domain of the mIL-4R followed by a transcripts are normally regulated in a parallel manshort C-terminal peptide sequence not present in the ner and further suggest that expression of both forms membrane form. of the IL-4R is controlled at multiple levels (i.e., tran-Whereas the function of the mIL-4R in ligand bindscriptional and posttranscriptional). ᭧ 1997 Academic Press ing and subsequent signaling appears obvious, the role of the sIL-4R is less clear. Since the sIL-4R binds IL-INTRODUCTION 4 with high affinity, it can act as a natural antagonist of IL-4 activity, competing with the mIL-4R on target Interleukin-4 (IL-4) is a pleiotropic cytokine procells for the binding of IL-4. Indeed, recombinant sILduced by the Th2 subset of CD4 / T cells, mast cells, 4R inhibits IL-4-dependent activities, both in vitro (21) and basophils (1, 2). Among its many activities are the and in vivo, in murine allograft rejection (22) and IgE activation and differentiation of B lymphocytes, includproduction after injection of anti-IgD antibodies (23) or ing the induction of immunoglobulin heavy-chain class allergens (24). Moreover, the administration of recombinant sIL-4R has also been reported to confer resistance in otherwise susceptible BALB/c mice against in-1 To whom correspondence and reprint requests should be addressed.
Specific antagonism of type I IL-4 receptor with a mutated form of murine IL-4
Journal of immunology (Baltimore, Md. : 1950), 1998
IL-4 is a pleiotropic cytokine that is essential for the differentiation of Th2 cells and is critically involved in the pathogenesis of certain infectious and allergic diseases. We have produced and functionally characterized a mutant of murine IL-4 (IL-4.Y119D) as a potential antagonist of IL-4. The analysis of IL-4R binding revealed no differences between wild-type and mutated IL-4. Despite this finding, IL-4.Y119D was unable to induce proliferation of several IL-4-responsive T cell lines mediated via the type I IL-4R (IL-4Ralpha/common gamma chain (gamma c chain)) and specifically inhibited the proliferative effect of wild-type IL-4. In contrast, with IL-4.Y119D we found induction of MHC class II and CD23 molecules on resting splenic B cells as well as proliferation of B9 plasmocytoma cells. In addition, IL-4.Y119D induced mRNA for soluble IL-4R, leading to the release of soluble IL-4R protein by spleen cells. In macrophages, mutated IL-4 in combination with IFN-gamma induced TNF...
Deletion of IL-4Rα on CD4 T Cells Renders BALB/c Mice Resistant to Leishmania major Infection
PLoS Pathogens, 2007
Effector responses induced by polarized CD4 þ T helper 2 (Th2) cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor a chain (IL-4Ra). IL-4Radeficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4 þ T cells and IL-4/IL-13 responsiveness of non-CD4 þ T cells in inducing nonhealer or healer responses have yet to be elucidated. CD4 þ T cell-specific IL-4Ra (Lck cre IL-4Ra À/lox ) deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Ra signaling during cutaneous leishmaniasis in the absence of IL-4-responsive CD4 þ T cells. Efficient deletion was confirmed by loss of IL-4Ra expression on CD4 þ T cells and impaired IL-4-induced CD4 þ T cell proliferation and Th2 differentiation. CD8 þ , cd þ , and NK-T cells expressed residual IL-4Ra, and representative non-T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Ra À/lox BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck cre IL-4Ra À/lox mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck cre IL-4Ra À/lox mice correlated with reduced numbers of IL-10-secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-c production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4 þ T cells is required to transform nonhealer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Ra signaling in L. major infection is revealed in which IL-4/IL-13-responsive non-CD4 þ T cells induce protective responses.
Quantitation of IL-4 expression in small numbers of cells from mice
Journal of Immunological Methods, 1993
Interleukin-4 (IL-4) is an important T cell and mast cell product that participates in allergic and cytotoxic responses, as well as functions as a growth factor for B, T, and inflammatory cells. Studies of the expression of IL-4 by T cells present in inflammatory reactions would be facilitated by using polymerase chain reaction (PCR) coupled to reverse transcription of mRNA to amplify the small quantity of mRNA present in these cells. In order to use this method in a quantitative manner, a plasmid was constructed that contained a modified form of mouse IL-4 cDNA. This plasmid was transcribed to produce cRNA for this modified sequence. The cRNA was used as an internal standard for the reverse transcription and amplification of IL-4 transcripts in RNA samples from mouse thymocytes. Amplification of reverse-transcribed native IL-4 mRNA produced a 286 bp PCR product. Amplification of the reverse-transcribed standard RNA produced a 155 bp product, which reflected a deletion introduced into the original IL-4 cDNA sequence. Comparison of the amount of the 286 bp native product to the amount of 155 bp standard product enabled the quantitative determination of IL-4 expression in each sample. This method was used to demonstrate that platelet activating factor increases the expression of IL-4 in mouse thymocytes and in a mouse T cell line. The expression of IL-4 by thymocytes exposed to platelet-activating factor (PAF) may reveal an important link between inflammation and the maturation of T cells in the thymus.
The Journal of Immunology
It was found that freshly isolated BALB/c CD4+ T cells produced high levels of IL-4 and IL-10 in response to immobilized anti-CD3 mAb, while C57BL/6 CD4+ T cells produced low amounts of IL-4 and IL-10. The high IL-4-producing ability of BALB/c mice was demonstrated to be genetically dominant and it was controlled by non-MHC gene (or genes). The cells responsible for IL-4 production in BALB/c mice were defined as TCRVp8.2+CD4+CD62L-CD45RB-memory-type T cells, which were distinct from NK1.l +CD4+NKT cells. Although these memory-type T cells were also detected in C57BL/6 mouse spleen at the same frequency, they showed a functionally different property from BALB/c CD4+CD62L-CD45RB-T cells in terms of IL-4 production. The fact that germfree BALB/c mouse spleen cells also produced high levels of IL-4 suggested that the 11-4 producer in BALB/c mice might be developed under the influence of unknown factors other than environmental Ags. The CD4+CD62L-CD45RB-T cells obtained from BALB/c mice accelerated the development of IL-4-producing memory-type CD4+ T cells from CD4+CD62L+CD45RB+ naive T cells prepared from OVA-specific TCR-transgenic mice. Therefore, IL-4producing CD4+CD62L-CD45RB-T cells might play an important role in the preferential induction of Th2-dominant immunity in BALB/c mouse strain.
Early IL-4 Production Does Not Predict Susceptibility toLeishmania major
Experimental Parasitology, 1996
mania major in mice can be self-limiting or fatal, depending upon the inbred mouse strain. It is well established that the outcome of infection is dependent upon the Th cell subset that dominates after infection. This has led to intense study of the early events associated with infection, in order to better understand the factors that determine Th1/2 cell development. In the present report, we have analyzed the kinetics of IL-4 and IL-4 mRNA production in three mouse strains: BALB/c, C3H, and C57BL/6. We found that in the first week IL-4 is absent in the C3H mice, but present in the susceptible BALB/c and relatively resistant C57BL/6 mouse. These data indicate that the presence of IL-4 by itself does not determine whether the immune response will be dominated by Th2 cells, since C57BL/6 mice will eventually develop a Th1 response and heal. We suggest that the critical cytokine that determines susceptibility in experimental leishmaniasis is IL-12, rather than IL-4. Thus, in C3H mice IL-12 is evident soon after infection, and IL-4 responses are not observed. In C57BL/6 mice, IL-12 production is delayed, but once evident, the IL-4 response is ablated. Further, we show that addition of IL-12 can block early IL-4 production in BALB/c mice, and neutralization of IL-12 in C3H mice uncovers IL-4 production in response to L. major infection. Taken together, these data indicate that susceptibility to L. major, while possibly requiring IL-4, is not determined by the presence or absence of IL-4.