Interleukin-11, an IL-6-like cytokine

The aim of this review is to collect various data on different functions of interleukin-11 (IL-11), a member of a family of IL-6-like cytokines. Numerous in vitro experiments have suggested a long list of IL-11 properties including support and control of proliferation and differentiation of hematopoietic progenitors, as well as participation in osteoclastogenensis, neurogenesis, and development of some other tissues. However, a great number of in vitro effects of IL-11 have not been confirmed in experiments using murine models, hampering an understanding of the physiological role of this cytokine. We discuss possible reasons for the divergence between in vitro and in vivo data as well as the perspectives of using conditional gene targeting to assess the role of IL-11 in ontogenesis and immune response.     

Human keratinocytes are major producers of IL-18: predominant expression of the unprocessed form

The cytokine network in the skin is a tightly regulated system in which IL-1 isoforms, as well as their receptors and antagonists have a central role. The recently discovered IL-1 isoform IL-18 (also known as interferon gamma-inducing factor (IGIF) or IL-1gamma), promotes IFN-gamma expression by T cells in concert with IL-12. Because IFN-gamma plays an important role in many inflammatory skin diseases by facilitating the development of Th1 cells, it is important to elucidate the role of mediators which regulate the production of this cytokine. We demonstrate that human keratinocytes constitutively express IL-18 at the mRNA as well as at the protein level. The protein was mainly expressed intracellularly in the 24 kD unprocessed pro-form, but was also secreted. Histochemistry revealed a diffuse staining of IL-18 in the epidermis of normal skin, which is in line with our in vitro data. Furthermore, we show that the level of IL-18 expressed in freshly isolated normal human epidermal cells, whether or not containing HLA-DR+ cells, significantly exceeded the expression levels of other cell types such as monocytes and bronchial epithelial cells. Finally, our results show that stimulation of the keratinocyte cell line HaCaT with PMA LPS or IL-1beta, does not significantly affect intracellular or released (pro) IL-18 levels. These experiments show for the first time that human keratinocytes relative to monocytes, PBMC or leukocytes produce a considerably larger amount of pro-IL-18, which is also readily released. High constitutive levels of IL-18 may contribute to the skewing towards a Th1-like environment, which is apparent in many human inflammatory skin diseases.     

Regulation of alloreactivity in vivo by IL-4 and the soluble IL-4 receptor.

Although numerous in vitro studies have demonstrated that cytokines are involved in the generation of alloreactive effector cells, the role of these regulatory substances in vivo is less well defined. We have recently cloned and expressed cDNAs encoding both membrane bound and soluble forms of the murine IL-4R. The effects of murine rIL-4 and a recombinant, soluble, extracellular portion of the murine IL-4R soluble(s) IL-4R on the generation of alloresponsiveness in vivo were evaluated by measuring the lymphoproliferative response to a localized injection of allogeneic cells and the survival of cardiac allografts. Administration of IL-4 to BALB/c mice resulted in a slight augmentation of the anti-C57BL/6 lymphoproliferative response compared to that occurring in control, mouse serum albumin-(MSA) treated mice. In contrast, the sIL-4R suppressed this response to allogeneic cells in a dose-dependent manner, with a dose of 50 micrograms/kg/day causing nearly complete inhibition of the response as compared to the response observed in controls. The inhibitory effect of sIL-4R was reversed by simultaneous administration of IL-4. A neutralizing antibody against IL-4 (11B11) and another against the IL-4R (M1) were also effective inhibitors of the response when given at 100- to 1000-fold higher concentrations than the amount of sIL-4R required to inhibit the response. In cardiac allograft experiments, BALB/c mice were engrafted with newborn C57BL/6 hearts in the ear pinnae and treated with sIL-4R (50 micrograms/kg) or MSA. Such allografts survived an average of 4 days longer in sIL-4R-treated mice than in MSA-treated controls. In conclusion, neutralization of IL-4 inhibits alloresponsiveness in vivo. These results confirm a regulatory role for this pleiotropic cytokine in allograft rejection and suggest a therapeutic value for IL-4 antagonists alone or in combination with other immunosuppressive regimens.     

Generation and immunologic functions of Th17 cells in malignant gliomas

Th17 cells, a recently discovered inflammatory T cell subtype, have been implicated with autoimmune disorders. However, mechanism of generation or functions of intratumoral Th17 cells are still unclear. We have been investigating the mechanism of induction and role of Th17 cells in malignant gliomas using primary tumor as well as cell lines. We report here that: (1) a higher frequency of Th17 cells in gliomas were associated with higher number of myeloid (CD11b) cells as well as the expression of TGF-β1 or IL-6; (2) conditioned medium from glioma cells (Gl CM) induced Th17 cell differentiation, which was inhibited by anti-TGF-β1 and anti-IL-6; (3) glioma-associated monocytes secreted Th17-promoting cytokines IL-1β and IL-23; (4) CM from glioma and monocyte co-culture (Gl+Mo CM) induced high frequency of Th17 cells in naïve T cell culture, which was abrogated by anti-IL-1β and anti-IL-23 antibodies; (5) In vitro Gl+Mo CM-mediated Th17 generation was associated with a decrease in IFN-γ and a concomitant increase in IL-10 secretion. Anti-TGF-β1, but not anti-IL-6, significantly reversed this cytokine profile. These results demonstrate prevalence of Th17 cells in gliomas and implicate the cytokines derived from the tumor as well as infiltrating myeloid cells in the induction of Th17 cells in glioma microenvironment. Moreover, the data also suggest that glioma-associated Th17 cells may contribute to immune-suppression via TGF-β1-induced IL-10 secretion. Further studies on the mechanism of tumor-infiltration, developmental pathways, and pro-/anti-tumor functions of Th17 cells will provide rationale for developing novel adjuvant immunotherapeutic strategies for malignant gliomas.     

Immune modulation in pemphigus vulgaris: role of CD28 and IL-10

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease characterized by Abs to the desmosomal cadherin desmoglein-3. Although the autoantibodies have been shown to be pathogenic, the role of the cellular immune system in the pathology of pemphigus-induced acantholysis is unclear. To further delineate the potential role of T cell-signaling pathways in the pathogenesis of PV, we performed passive transfer experiments with PV IgG in gene-targeted mutant mice. Our results demonstrated that CD28-deficient mice (lacking a costimulatory signal for T cell activation) are 5-fold more sensitive to the development of PV than wild-type mice. To evaluate whether the higher incidence of disease was due to an impairment in intercellular adhesion of keratinocytes, we performed an in vitro acantholysis, using CD28-/- mice keratinocytes. No alteration in in vitro adhesion was detected in CD28-/--type keratinocytes. Because the CD28 molecule plays a pivotal role in the induction of Th2 cytokines, we examined the levels of a prototypic Th2 cytokine (IL-10) in CD28-/- mice. Lower levels of IL-10 mRNA were found in lesions from CD28-/- mice. To determine whether pemphigus susceptibility in CD28-/- was related to IL-10 deficiency, we performed passive transfer experiments in IL-10-/- mice that demonstrated increased blisters compared with controls. To confirm that IL-10 is involved in the pathogenesis, rIL-10 was given with PV IgG. IL-10 significantly suppressed the disease activity. These data suggest a potential role of IL-10 in PV.     

Regulation of carcinogenesis by IL-5 and CCL11: a potential role for eosinophils in tumor immune surveillance

The role of the immune system in the surveillance of transformed cells has seen a resurgence of interest in the last 10 years, with a substantial body of data in mice and humans supporting a role for the immune system in host protection from tumor development and in shaping tumor immunogenicity. A number of earlier studies have demonstrated that eosinophils, when recruited into tumors, can very effectively eradicate transplantable tumors. In this study, we investigated whether eosinophils also play a role in tumor immune surveillance by determining the incidence of methylcholanthrene (MCA)-induced fibrosarcomas in IL-5 transgenic mice that have greatly enhanced levels of circulating eosinophils, CCL11 (eotaxin-1)-deficient mice that lack a key chemokine that recruits eosinophils into tissues, and the eosinophil-deficient mouse strains, IL-5/CCL11(-/-) and DeltadblGATA. It was found that MCA-induced tumor incidence and growth were significantly attenuated in IL-5 transgenic mice of both the BALB/c and C57BL/6 backgrounds. Histological examination revealed that the protective effect of IL-5 was associated with massively enhanced numbers of eosinophils within and surrounding tumors. Conversely, there was a higher tumor incidence in CCL11(-/-) BALB/c mice, which was associated with a reduced eosinophil influx into tumors. This correlation was confirmed in the eosinophil-deficient IL-5/CCL11(-/-) and DeltadblGATA mouse strains, where tumor incidence was greatly increased in the total absence of eosinophils. In addition, subsequent in vitro studies found that eosinophils could directly kill MCA-induced fibrosarcoma cells. Collectively, our data support a potential role for the eosinophil as an effector cell in tumor immune surveillance.     

The role of IL-5 in the immune responses to nematodes in rodents

In this review, Masataka Korenoga and Isoo Toda discuss the role of interleukin 5 (IL-5) in immune responses caused by parasitic infections. Recent studies have shown that eosinophilia in parasitic infections is dependent on IL-5. Although eosinophils have been thought to be potent effector cells (from results of experiments, in vitro), their role in protective immunity to parasites is controversial.     

Interleukin-10 is up-regulated by prolactin and serum-starvation in cultured mammary epithelial cells

The epithelial cells of the mammary gland go through a cycle of growth, differentiation, and involution during pregnancy. Recently, we found that interleukin-10 (IL-10) was induced at the involution stage and contributed to apoptosis in the mammary gland. To elucidate the role of the epithelial cells in involution, we examined IL-10 expression in an in vitro model of HC11 cells, in various culture conditions. IL-10 was weakly expressed early in growth but when the cells were induced to differentiate by insulin and dexamethasone expression increased slightly. Prolactin in combination with insulin and dexamethasone caused a further increase. To mimic apoptosis the culture was deprived of serum as well as hormones, and this resulted in a gradual increase in IL-10. In contrast with its ligand, the IL-10 receptor itself was not expressed in any conditions. We speculate that release of IL-10 from the epithelial cells recruits lymphocytes, which have IL-10 receptors on their cell membranes, and they in turn secrete death factors inducing apoptosis of the epithelial cells.     

IL-17 Enhances Chemotaxis of Primary Human B Cells during Asthma

IL-17 is a pro-inflammatory mediator that is believed to play a critical role in regulating tissue inflammation during asthma, COPD, as well as other inflammatory disorders. The level of expression of IL-17 has been shown to be upregulated in lung bronchial tissue of asthmatic patients. Several reports have provided further evidence that this cytokine could play a key role in enhancing the migration of inflammatory as well as structural cells of the bronchial lung tissue during asthma and COPD. B cell infiltration to sites of inflammation during inflammatory disorders such as bowel disease, asthma and COPD has been reported. Accordingly, in this study we hypothesized that IL-17 may exert a chemotactic effect on primary B cells during asthma. We observed that B cells from asthmatic patients expressed significantly higher levels of IL-17RA and IL-17RC, compared to those of healthy subjects. Using an in-vitro migration assay, B cells were shown to migrate towards both IL-17A and IL-17F. Interestingly, blocking IL-17A and IL-17F signaling using either anti-IL-17R antibodies or MAP kinase inhibitors prevented in vitro migration of B cell towards IL-17. These observations indicate a direct chemotactic effect of IL-17 cytokines on primary peripheral blood B cells with higher effect being on asthmatic B cells. These findings revealed a key role for IL-17 in enhancing the migration of B cells to the lung tissue during asthma or COPD.     

Interleukin-11 signals through the formation of a hexameric receptor complex

Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. IL-11 has been shown to induce gp130-dependent signaling through the formation of a high affinity complex with the IL-11 receptor (IL-11R) and gp130. Site-directed mutagenesis studies have identified three distinct receptor binding sites of IL-11, which enable it to form this high affinity receptor complex. Here we present data from immunoprecipitation experiments, using differentially tagged forms of ligand and soluble receptor components, which show that multiple copies of IL-11, IL-11R, and gp130 are present in the receptor complex. Furthermore, it is demonstrated that sites II and III of IL-11 are independent gp130 binding epitopes and that both are essential for gp130 dimerization. We also show that a stable high affinity complex of IL-11, IL-11R, and gp130 can be resolved by nondenaturing polyacrylamide gel electrophoresis, and its composition verified by second dimension denaturing polyacrylamide gel electrophoresis. Results indicate that the three receptor binding sites of IL-11 and the Ig-like domain of gp130 are all essential for this stable receptor complex to be formed. We therefore propose that IL-11 forms a hexameric receptor complex composed of two molecules each of IL-11, IL-11R, and gp130.     

Endogenous IL-11 is pro-inflammatory in acute methylated bovine serum albumin/interleukin-1-induced (mBSA/IL-1)arthritis

OBJECTIVE: To evaluate the role of interleukin-11 (IL-11) in acute mBSA/IL-1-induced inflammatory arthritis. METHODS: IL-11 was administered via intra-articular (IA) injection into knee joints of C57BL/6 mice and joint histology was assessed. The mitogenic response to IL-11 was measured in wild-type (WT) synovial fibroblasts. IL-1 was used as a comparator in both the studies. The severity of acute methylated bovine serum albumin (mBSA)/IL-1 arthritis was determined in WT and IL-11 receptor null (IL-11Ra1-/-) mice. In parallel experiments, a neutralising antibody to IL-11 was administered to WT mice throughout this model. RESULTS: IA injections of IL-11 resulted in mild-to-moderate joint inflammation which was less than that due to IA IL-1. IL-11 had a dose-dependent mitogenic effect on WT synovial fibroblasts (P<0.01). mBSA/IL-1 acute arthritis was reduced in IL-11Ra1-/- versus WT mice (histological arthritis score: 10.1+/-0.5 versus 12.8+/-0.7, respectively; P=0.01). Administration of an IL-11 neutralising antibody to WT mice reduced mBSA/IL-1 acute arthritis scores compared to control antibody (10.6+/-0.7 versus 13.3+/-0.6, respectively; P=0.02). CONCLUSIONS: These data demonstrate that endogenous IL-11 exerts relatively mild but consistent pro-inflammatory effects in acute inflammatory arthritis.     

Differential expression of CD11c by peripheral blood NK cells reflects temporal activity of multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease, showing a great degree of variance in temporal disease activity. We have recently demonstrated that peripheral blood NK cells biased for secreting IL-5 (NK2 bias) are associated with the remission state of MS. In this study, we report that MS patients in remission differentially express CD11c on NK cell surface (operationally defined as CD11chigh or CD11clow). When we compared CD11chigh or CD11clow patients, the expression of IL-5 and GATA-3 in NK cells supposed to endow a disease-protective NK2 phenotype was observed in CD11clow but not in CD11chigh patients. In contrast, the CD11chigh group showed a higher expression of HLA-DR on NK cells. In vitro studies demonstrated that NK cell stimulatory cytokines such as IL-15 would up-regulate CD11c expression on NK cells. Given previous evidence showing an association between an increased level of proinflammatory cytokines and temporal disease activity in MS, we postulate that inflammatory signals may play a role in inducing the CD11chigh NK cell phenotype. Follow-up of a new cohort of patients showed that 6 of 10 CD11chigh MS patients developed a clinical relapse within 120 days after evaluation, whereas only 2 of 13 CD11clow developed exacerbated disease (p = 0.003). As such, a higher expression of CD11c on NK cells may reflect the temporal activity of MS as well as a loss of regulatory NK2 phenotype, which may allow us to use it as a potential biomarker to monitor the immunological status of MS patients.     

Role of interleukin-1 in 4-hydroperoxycyclophosphamide toxicity to bone marrow progenitor cells: A review

We have demonstrated that in vitro preincubation with IL-1 or TNFa for 20 hours can protect human hematopoietic progenitors from lethal doses of 4-HC. On the other hand, preincubation with IL-6 or IL-3, in a similar fashion, did not provide any protection but in fact demonstrated a slight increase in 4-HC toxicity in the same experiments. The observation that IL-1 was still protective even when a purified cell population depleted of accessory cells was used is suggestive of a direct effect of IL-1. Our data also suggest that early progenitor cells including the replatable B;-CFC are the main target of that protection. We believe that using this in vitro assay system will enable us to investigate the possible mechanisms responsible for the protection of these primitive progenitors. From a clinical perspective, future studies should attempt to clarify whether protection by IL-1 is selective for normal hematopoietic cells versus malignant cells and whether these protected primitive progenitors represent the pluripotent stem cells responsible for engraftment of transplanted bone marrow by using an animal model system.     

Interleukin-1 mediates endothelin-1-induced fever and prostaglandin production in the preoptic area of rats

The intracerebroventricular injection of endothelin-1 (ET-1) induces fever and increases PG levels in the cerebrospinal fluid of rats. Likewise, the injection of IL-1 into the preoptic area (POA) of the rat hypothalamus causes both fever and increased PG production. In this study, we conducted in vivo and in vitro experiments in the rat to investigate 1) the hypothalamic region involved in ET-1-induced fever and PG biosynthesis and 2) whether hypothalamic IL-1 plays a role as a mediator of the above ET-1 activities. One hundred femtomoles of ET-1 increased body temperature when injected in the POA of conscious Wistar rats; this effect was significantly counteracted by the coinjection of 600 pmol IL-1 receptor antagonist (IL-1ra). In experiments on rat hypothalamic explants, 100 nM ET-1 caused a significant increase in PGE2 production and release from the whole hypothalamus and from the isolated POA, but not from the retrochiasmatic region, in 1-h incubations. Six nanomoles of IL-1ra or 10 nM of a cell-permeable interleukin-1 converting enzyme inhibitor completely counteracted the effect of ET-1 on PGE2 release from the POA. One hundred nanomoles ET-1 also caused a significant increase in IL-1beta immunoreactivity released into the bath solution of hypothalamic explants after 1 h of incubation, although during such time ET-1 failed to modify the gene expression of IL-1beta and other pyrogenic cytokines within the hypothalamus. In conclusion, our results show that ET-1 increases IL-1 production in the POA, and this effect appears to be correlated to ET-1-induced fever in vivo, as well as to PG production in vitro.     

Migratory activity of human breast cancer cells is modulated by differential expression of xanthine oxidoreductase

Xanthine oxidoreductase (XOR) may exert an important, but poorly defined, role in the pathogenesis of breast cancer (BC). Loss of XOR expression was linked to aggressive BC, and recent clinical observations have suggested that decreasing XOR may be functionally linked to BC aggressiveness. The goal of the present investigation was to determine whether the decreased XOR observed in clinically aggressive BC was an intrinsic property of highly invasive mammary epithelial cells (MEC). Expression of XOR was investigated using HC11 mouse MEC, HB4a and MCF-10A normal human MEC, and several human mammary tumor cells including MCF-7 and MDA-MB-231. Consistent with clinical observations, data shown here revealed high levels of XOR in normal HC11 and MCF-10A cells that was markedly reduced in highly invasive mammary tumor cells. The contribution of XOR to tumor cell migration in vitro was investigated using MDA-MB-231 and MCF-7 cells and clonally selected derivatives of HC11 that exhibit either weak or strong migration in vitro. We observed that over-expression of an XOR cDNA in MDA-MB-231 and in HC11-C24, both possessing weak XOR expression and high migratory capacity, inhibited their migration in vitro. Conversely, pharmacological inhibition of XOR in MCF-7 and HC11-C4, both possessing high XOR expression and weak migratory capacity, stimulated their migration in vitro. Further experiments suggested that XOR derived ROS mediated this effect and also modulated COX-2 and MMP levels and function. These data demonstrate a functional link between XOR expression and MEC migration and suggest a potential role for XOR in suppressing BC pathogenesis.     

Chlamydia infection induces ICOS ligand-expressing and IL-10-producing dendritic cells that can inhibit airway inflammation and mucus overproduction elicited by allergen challenge in BALB/c mice

Our previous study has shown that the adoptive transfer of dendritic cells (DCs) freshly isolated from Chlamydia-infected mice (iIDCs), unlike those from control naive mice (iNDCs), can inhibit systemic and cutaneous eosinophilia induced by OVA exposure. In the present study, we examined the mechanism by which iIDC inhibits allergen-specific Th2 cell differentiation in vitro and in vivo. The study revealed that iIDCs exhibited higher surface expression of CD8alpha and the ICOS ligand (ICOS-L), as well as higher IL-10 and IL-12 production than iNDCs. In vitro DC:CD4(+) T cell coculture experiments showed that iIDCs could inhibit allergen-specific Th2 cell differentiation and that the inhibitory effect could be abolished by the blockage of IL-10 or IL-12 activity. More interestingly, the coblockade of IL-10 and the ICOS-L showed synergistic effect in enhancing allergen-driven Th2 cytokine production. Furthermore, adoptive transfer of iIDCs, but not iNDCs, to OVA sensitized mice significantly inhibited airway eosinophilia and mucus overproduction following intranasal challenge with OVA. Overall, the data demonstrate a critical role played by ICOS-L-expressing and IL-10-producing DCs from Chlamydia-infected mice in the infection-mediated inhibition of allergic responses.     

Adoptive transfer of CD8alpha+ dendritic cells (DC) isolated from mice infected with Chlamydia muridarum are more potent in inducing protective immunity than CD8alpha- DC

Chlamydial infections are serious public health concerns worldwide. In this study, we examined the role of dendritic cell (DC) subsets in inducing protective immunity against chlamydial infection using an adoptive transfer approach. We found that CD11c+CD8alpha+ (double-positive, DP) DC, compared with CD11c+CD8alpha- (single-positive, SP) DC isolated from infected mice, are more potent inducers of protective immunity. Specifically, mice pretreated with DPDC from infected mice, upon infection with Chlamydia trachomatis mouse pneumonitis (MoPn), experienced significantly less severe body weight loss and in vivo chlamydial growth. Analysis of MoPn-driven cytokine production by immune cells revealed that mice that were treated with DPDC produced significantly higher levels of Th1 (TNF-alpha, IFN-gamma, and IL-12) but lower levels of Th2 (IL-4, IL-5, and IL-13)-related cytokines than the recipients of SPDC following infection challenge. Moreover, DPDC-treated mice displayed significantly higher levels of MoPn-specific IgG2a production and delayed-type hypersensitivity responses compared with SPDC-treated mice. Furthermore, DPDC isolated from infected mice produced higher amounts of IL-12 and IL-10 in vitro in comparison with SPDC. These data indicate that CD8alpha+ DC have a significantly higher capacity in inducing protective immunity compared with CD8alpha- DC, demonstrating the crucial role of DC1-like cells in eliciting protection against C. trachomatis infection.