The role of interleukin-5 (IL-5 ) in vivo: studies with IL-5 deficient mice

Eosinophil recruitment is a characteristic feature of a number of pathological conditions and was the topic of the recent International Symposium on allergic inflammation, asthma, parasitic and infectious diseases (Rio de Janeiro, June 3-5, 1996). Since interleukin5 (IL5) is believed to regulate the growth, differentiation and activation of eosinophils (Coffman et al. 1989, Sanderson 1992), the role of eosinophils and IL5 are closely linked. Although IL5 specifically regulates eosinophilia in vivo and this is its most well established activity, it is becoming clear that IL5 also has other biological effects. The recent derivation of an IL5 deficient mouse (Kopf et al. 1996), provides a model for exploring not only the role of IL5 and eosinophils but also other novel activities of IL5. Of note is that although the IL5 deficient mice cannot elicit a pronounced eosinophilia in response to inflammatory stimulation following aeroallergen challenge or parasite infection they still produce basal levels of eosinophils that appear to be morphologically and functionally normal. However, the basal levels of eosinophils appear insufficient for normal host defence as IL5 deficiency has now been shown to compromise defence against several helminth infections. In addition, IL5 deficient mice appear to have functional deficiencies in B-1 B lymphocytes and in IgA production.

Activation of CD4+ T lymphocytes form interleukin 2-deficient mice by costimulatory B7 molecules.

Interleukin 2 (IL-2)-deficient (IL-2-/-) mice develop hemolytic anemia and chronic inflammatory bowel disease. Importantly, the induction of disease in IL-2-deficient mice is critically dependent on CD4+ T cells. We have studied the requirements of T cells from IL-2-deficient mice for costimulation with B7 antigens. Stable B7-1 or B7-2 chinese hamster ovary (CHO) cell transfectants could synergize with anti-CD3 monoclonal antibody (mAb) to induce the proliferation of CD4+ T cells from IL-2-/- mutant mice. Further mechanistic studies established that B7-induced activation resulted in surface expression of the alpha chain of the IL-2 receptor. B7-induced proliferation occurred independently of IL-4 and was largely independent of the common gamma chain of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors. Finally, anti-B7-2 but not anti-B7-1 mAb was able to inhibit the activation of IL-2-/- T cells induced by anti-CD3 mAb in the presence of syngeneic antigen-presenting cells. The results of our experiments indicate that IL-2-/- CD4+ T cells remain responsive to B7 stimulation and raise the possibility that B7 antagonists have a role in the prevention/treatment of inflammatory bowel disease.

Interleukin-6-deficient mice are highly susceptible to Giardia lamblia infection but exhibit normal intestinal immunoglobulin A responses against the parasite.

In the present study, interleukin-6 (IL-6)-deficient mice were infected with Giardia lamblia clone GS/M-83-H7. Murine IL-6 deficiency did not affect the synthesis of parasite-specific intestinal immunoglobulin A. However, in contrast to wild-type mice, IL-6-deficient animals were not able to control the acute phase of parasite infection. Reverse transcription-PCR-based quantitation of cytokine mRNA levels in peripheral lymph node cells exhibited a short-term up-regulation of IL-4 expression in IL-6-deficient mice that seemed to be associated with failure in controlling the parasite population. This observation suggests a further elucidation of IL-4-dependent, Th2-type regulatory processes regarding their potential to influence the course of G. lamblia infection in the experimental murine host.

Altered hypertrophic chondrocyte kinetics in GDF-5 deficient murine tibial growth plates

The growth/differentiation factors (GDFs) are a subgroup of the bone morphogenetic proteins best known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling proteins, GDF-5, exhibit numerous skeletal abnormalities, including shortened limb bones. The primary aim of this study was determine whether GDF-5 deficiency would alter the growth rate in growth plates from the long bones in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5-week-old female GDF-5 -/- mice and control littermates were examined. GDF-5 deficiency resulted in a statistically significant reduction in growth rate (-14%, p=0.03). The effect of genotype on growth rate was associated with an altered hypertrophic phase duration, with hypertrophic cells from GDF-5 deficient mice exhibiting a significantly longer phase duration compared to control littermates (+25%, p=0.006). These data suggest that one way in which GDF-5 might modulate the rate of endochondral bone growth could be by affecting the duration of the hypertrophic phase in growth plate chondrocytes.

The enigmatic eosinophil: investigation of the biological role of eosinophils in parasitic helmint infection

In many helminth infected hosts the number of eosinophils increases dramatically, often without any concurrent increases in the number of other leukocytes, so that eosinophils become the dominant cell type. Many experimental investigations have shown that the eosinophilia is induced by interleukin-5 (IL-5) but its functional significance remains unclear. Mice genetically deficient in IL-5 (IL-5-/-) have been used to evaluate the functional consequences of the IL-5 dependent eosinophilia in helminth infected hosts. Host pathology and level of infection were determined in IL-5-/- and wild type mice infected with a range of species representative of each major group of helminths. The effects of IL-5 deficiency were very heterogeneous. Of the six species of helminth examined, IL-5 dependent immune responses had no detectable effect in infections with three species, namely the cestodes Mesocestoides corti and Hymenolepis diminuta and the trematode Fasciola hepatica. In contrast, IL-5 dependent immune responses were functionally important in mice infected with three species, notably all nematodes. Damage to the lungs caused by migrating larvae of Toxocara canis was reduced in IL-5-/- mice. Infections of the intestine by adult stages of either Strongyloides ratti or Heligmosomoides polygyrus were more severe in IL-5-/- mice. Adult intestinal nematodes were clearly deleteriously affected by IL-5 dependent processes since in its presence there were fewer worms which had reduced fecundity and longevity. The implications of these results for the viability of using inhibitors of IL-5 as a therapy for asthma are considered.

Leishmania braziliensis: partial control of experimental infection by interleukin-12 p40 deficient mice

Resistance to infection by Leishmania major has been associated with the development of a Th1 type response that is dependent on the presence of interleukin 12 (IL-12). In this work the involvement of this cytokine in the response to infection by L. braziliensis, a less virulent species in the mouse model, was evaluated. Our results show that while interferon (IFN-g) deficient (-/-) mice inoculated L. braziliensis develop severe uncontrolled lesions, chronic lesions that remained under control up to 12 weeks of infection were observed in IL-12p40 -/- mice. IL 12p40 -/- mice had fewer parasites in their lesions than IFN-g-/- mice. Lymph node cells from IL-12p40 -/- were capable of producing low but consistent levels of IFN-g suggestive of its involvement in parasite control. Furthermore, as opposed to previous reports on L. major-infected animals, no switch to a Th2 response was observed in IL-12p40 -/- infected with L. braziliensis.

Preliminary results on interleukin-4 and interleukin-10 cytokine production in malnourished, inducible nitric oxide synthase-deficient mice with schistosomiasis mansoni infection

Schistosoma mansoni infected C57Bl/6 inducible nitric oxide synthase (iNOS)-deficient and non-deficient malnourished mice, both fed a balanced controlled diet were studied. Interleukins, IL-4 and IL-10 responses to soluble egg antigens (SEA) 90 days after infection, were determined. Our results suggest that in iNOS deficient, malnourished mice, 90 days after of infection, nitric oxide has a downregulating effect on IL-4 and IL-10 production. We are currently investigating the biological significance of these findings.

Regulation of the cardiac sodium channel Nav1.5 by utrophin in dystrophin-deficient mice

Duchenne muscular dystrophy (DMD) is a severe striated muscle disease due to the absence of dystrophin. Dystrophin deficiency results in dysfunctional sodium channels and conduction abnormalities in hearts of mdx mice. Disease progression in the mdx mouse only modestly reflects that of DMD patients, possibly due to utrophin up-regulation. Here, we investigated mice deficient in both dystrophin and utrophin [double knockout (DKO)] to assess the role of utrophin in the regulation of the cardiac sodium channel (Na(v)1.5) in mdx mice.

A critical role of Lyn and Fyn for B cell responses to CD38 ligation and interleukin 5

CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induced proliferation, interleukin 5 (IL-5) receptor α chain expression, and tyrosine phosphorylation of Bruton tyrosine kinase (Btk) from wild-type, but not from X chromosome-linked, immunodeficient mice. B cells from fyn-deficient (Fyn−/−) and lyn-deficient (Lyn−/−) mice showed an impaired response to mAb CS/2 for proliferation and IL-5 receptor α chain expression, and B cells from fyn/lyn double-deficient (Fyn/Lyn−/−) mice did not respond at all to mAb CS/2. The Btk activation by CD38 ligation was observed in B cells from Fyn−/− mice, and it was severely impaired in B cells from Lyn−/− and Fyn/Lyn−/− mice. CD38 expression on B cells from three mutant strains was comparable to that on control B cells. We infer from these results that both Fyn and Lyn are required and that their signals are synergistic for B cell triggering after CD38 ligation. Lyn is upstream of Btk activation in the CD38 signaling. Stimulation of B cells with IL-5 together with CD38 ligation induces not only IgM but also IgG1 secretion. Analysis of the synergistic effects of IL-5 and CD38 ligation on IgG1 secretion revealed the impaired IgG1 secretion of B cells from Lyn−/− and Fyn/Lyn−/− mice. These data imply that Lyn is involved in B cell triggering by CD38 ligation plus IL-5 for isotype switching.

An interleukin 4 (IL-4)-independent pathway for CD4+ T cell IL-4 production is revealed in IL-4 receptor-deficient mice

IL-4 receptor α chain (IL-4Rα)-deficient mice were generated by gene-targeting in BALB/c embryonic stem cells. Mutant mice showed a loss of IL-4 signal transduction and functional activity. The lack of IL-4Rα 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-4Rα−/− 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-4Rα−/−, β2-microglobulin−/− double homozygous mice produced less IL-4 than did either IL-4Rα−/− or wild-type mice. These results indicate that an IL-4-independent, β2-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.

Interleukin 7 receptor-deficient mice lack gammadelta T cells.

The interleukin 7 receptor (IL-7R) plays a crucial role in early B- and T-cell development. It consists of a unique a chain and a common gamma chain [IL-2 receptor gamma chain (IL-2Rgamma)]. Gene inactivation of IL-7, IL-7R, and IL-2Rgamma resulted in severe impairment of B and T lymphopoiesis in mice. In addition, IL-2Rgamma-deficient mice lack gammadelta T cells in the skin and have the impaired development of natural killer (NK) cells and intraepithelial lymphocytes. To explore the role of IL-7/IL-7R system in gammadelta T- and NK-cell development, we have generated and analyzed IL-7R-deficient mice. gammadelta T cells were absent from skin, gut, liver, and spleen in the deficient mice. In contrast, alphabeta T and B cells were detected in reduced, but certain, numbers, and NK cells developed normally. The gammadelta T-cell development in fetal and adult thymus was also completely blocked. These results clearly demonstrate that the signal from IL-7R is indispensable for gammadelta T-cell development in both thymic and extrathymic pathways. On the contrary, it is suggested that NK-cell development requires cytokine(s) other than IL-7.

Eosinophil recruitment into the respiratory epithelium following antigenic challenge in hyper-IgE mice is accompanied by interleukin 5-dependent bronchial hyperresponsiveness.

A murine model for antigen-induced bronchial hyperreactivity (BHR) and airway eosinophilia, two hallmarks of asthma, was developed using ovalbumin-immunized mice, which produce large amounts of IgE (named BP2, "Bons Producteurs 2," for High Line of Selection 2). A single intranasal ovalbumin challenge failed to modify the bronchial responses, despite the intense eosinophil recruitment into the bronchoalveolar lavage fluid and airways. When mice were challenged twice a day for 2 days or once a day for 10 days, BHR in response to i.v. 5-hydroxytryptamine or to inhaled methacholine was induced in BP2 mice but not in BALB/c mice. Histological examination showed that eosinophils reached the respiratory epithelium after multiple ovalbumin challenges in BP2 mice but remained in the bronchial submucosa in BALB/c mice. Total IgE titers in serum were augmented significantly with immunization in both strains, but much more so in BP2 mice. Interleukin 5 (IL-5) titers in serum and bronchoalveolar lavage fluid of BP2 mice were augmented by the antigenic provocation, and a specific anti-IL5 neutralizing antibody suppressed altogether airway eosinophilia and BHR, indicating a participation of IL-5 in its development. Our results indicate that the recruitment of eosinophils to the airways alone does not induce BHR in mice and that the selective effect on BP2 mice is related to their increased IgE titers associated with antigen-driven eosinophil migration to the epithelium, following formation and secretion of IL-5.

Immunolocalization and pathological alterations following Strongyloides venezuelensis infection in the lungs and the intestine of MHC class I or II deficient mice

The present study, investigated the mechanisms involved in the immune responses of Major Histocompatibility Complex class I or class II knockout mice, following Strongyloides venezuelensis infection. Wild-type C57BL/6 (WT), MHC II(-/-) and MHC I(-/-) mice were individually inoculated with 3000 larvae (U) of S. venezuelensis and sacrificed on days 1, 3, 5, 8, 13 and 21 post-infection (p.i.). Samples of blood, lungs and small intestines were collected. The tissue samples were stained with hematoxylineosin for the pathological analysis. The presence of the parasite was demonstrated by immunoperoxidase analysis. MHC II(-/-) mice presented a significantly higher number of adult worms recovered from the small intestine on day 5 p.i. and presented elevated numbers of eggs in the feces. The infection by S. venezuelensis was completely eliminated 13 days after infection in WT as well as in MHC I(-/-) mice. In MHC II(-/-) mice, eggs and adult worms were still found on day 21 p.i., however, there was a significant reduction in their numbers. In the lung, the parasite was observed in MHC I(-/-) on day 1 p.i. and in MHC II(-/-) mice on days 1 and 5 p.i. In the small intestine of WT mice, a larger number of parasites were observed on day 8 p.i. and their absence was observed after day 13 p.i. Through immunohistochemistry analysis, the parasite was detected in the duodenum of WT on days 5 and 8 p.i., and in knockout mice on days 5, 8 and 13 p.i.; as well as in posterior portions of the small intestine in MHC I(-/-) and MHC II(-/-) on day 13 p.i., a finding which was not observed in WT mice. We concluded that immunohistochemistry analysis contributed to a more adequate understanding of the parasite localization in immunodeficient hosts and that the findings aid in the interpretation of immunopathogenesis in Strongyloides infection. (C) 2008 Elsevier B.V. All rights reserved.

Morphological and Molecular Pathology of CCl(4)-Induced Hepatic Fibrosis in Connexin43-Deficient Mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4)-induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech. 74:421-429, 2011. (C) 2010 Wiley-Liss, Inc.