Partners in crime: VEGF and IL-4 conscript tumour-promoting macrophages

Tumour-associated macrophages (TAMs) foster tumour progression by several mechanisms, including the promotion of angiogenesis, tissue remodelling, and immunosuppression. Such pro-tumoural activities are thought to be executed by TAM subtypes that harbour features of alternatively activated (or M2-polarized) macrophages. However, the molecular signals in tumours that induce recruitment and differentiation of M2-like macrophages are not fully defined. In this issue of The Journal of Pathology, Linde et al investigate the role of the tumour-derived cytokines, VEGF and IL-4, in the recruitment and polarization of macrophages in a mouse model of skin cancer. The authors report that while VEGF-A recruits monocytes from the peripheral circulation, IL-4 induces their differentiation into tumour-promoting, M2-like macrophages. IL-4 signalling blockade was sufficient to reprogram TAMs away from the M2-like phenotype and inhibited tumour angiogenesis and growth. This study attests to the potential of reprogramming TAMs to abate their pro-angiogenic and pro-tumoural functions in tumours.     

Possible involvement of the M2 anti-inflammatory macrophage phenotype in growth of human gliomas

Within tumours, many non-neoplastic cells such as fibroblasts, endothelial cells, and macrophages assist tumour growth by producing various growth factors and pro-angiogenic cytokines. Various tumour-derived molecules drive tumour-associated macrophages towards an anti-inflammatory phenotype (M2) and thus promoting tumour growth. Here we investigated microglia/macrophage differentiation in glioma tissues by means of immunostaining of paraffin-embedded glioma samples. The number of microglia/macrophages with positive staining for CD163 and CD204, which are believed to be markers for M2 macrophages, was correlated with the histological grade of the gliomas. The ratio of M2 macrophages in the tumour-associated microglia/macrophages was also associated with the histological grade. Culture supernatant from the glioma cell line can stimulate macrophages to develop into the M2 phenotype in vitro. Macrophage colony-stimulating factor (M-CSF), which strongly induces M2 polarization of macrophages, was significantly correlated with histological malignancy and with the proportion of M2 microglia/macrophages in vivo. In addition, the proportion of M2 microglia/macrophages and M-CSF expression in tumour cells correlated well with proliferation of glioblastoma cells. These results suggest that tumour-derived M-CSF induces a shift of microglia/macrophages towards the M2 phenotype, which influences tumour growth. Evaluation of the proportion of M2 microglia/macrophages and M-CSF expression in tumour tissue would be useful for assessment of microglia/macrophage proliferative activity and the prognosis of patients with gliomas.     

Diffuse large B cell lymphoma-derived extracellular vesicles educate macrophages to promote tumours progression by increasing PGC-1β

Tumour-associated macrophages (TAMs) comprise an important part of the tumour microenvironment and play a key role in malignant tumours progression. Tumour-derived extracellular vesicles (EVs) modulated TAMs polarization and reprogrammed TAMs to influence the progression of various tumours. Here, we hypothesized that diffuse large B cell lymphoma (DLBCL)-derived EVs can regulate macrophages polarization and thus contribute to tumour progression. Our results demonstrated that EVs, released from DLBCL, augment the M2 polarization effects of macrophages. Moreover, conditional medium derived from macrophages by DLBCL-derived EVs stimulation revealed the superior effects of promoting tumour proliferation, migration and invasion. Further analysis demonstrated that DLBCL-derived EVs regulated the metabolism of macrophages by increasing the expression of PGC-1β protein, thereby reprogramming the macrophage phenotype of promoting tumour progression. In conclusion, our findings signify that the DLBCL-derived EVs mediated the increasing of functional PGC-1β protein in macrophages to promote the tumour progression.     

VAP-1-mediated M2 macrophage infiltration underlies IL-1β- but not VEGF-A-induced lymph- and angiogenesis

Vascular adhesion protein-1 (VAP-1) contributes to inflammatory and angiogenic diseases, including cancer and age-related macular degeneration. It is expressed in blood vessels and contributes to inflammatory leukocyte recruitment. The cytokines IL-1β and vascular endothelial growth factor A (VEGF-A) modulate angiogenesis, lymphangiogenesis, and leukocyte infiltration. The lymphatic endothelium expresses intercellular adhesion molecule-1 and vascular adhesion molecule-1, which facilitate leukocyte transmigration into the lymphatic vessels. However, whether lymphatics express VAP-1 and whether they contribute to cytokine-dependent lymph- and angiogenesis are unknown. We investigated the role of VAP-1 in IL-1β- and VEGF-A-induced lymph- and angiogenesis using the established corneal micropocket assay. IL-1β increased VAP-1 expression in the inflamed cornea. Our in vivo molecular imaging revealed significantly higher VAP-1 expression in neovasculature than in the preexisting vessels. VAP-1 was expressed in blood but not lymphatic vessels in vivo. IL-1β-induced M2 macrophage infiltration and lymph- and angiogenesis were blocked by VAP-1 inhibition. In contrast, VEGF-A-induced lymph- and angiogenesis were unaffected by VAP-1 inhibition. Our results indicate a key role for VAP-1 in lymph- and angiogenesis-related macrophage recruitment. VAP-1 might become a new target for treatment of inflammatory lymph- and angiogenic diseases, including cancer.     

Distinct roles of CSF family cytokines in macrophage infiltration and activation in glioma progression and injury response

Gliomas attract brain‐resident (microglia) and peripheral macrophages and reprogram these cells into immunosuppressive, pro‐invasive cells. M‐CSF (macrophage colony‐stimulating factor, encoded by the CSF1 gene) has been implicated in the control of recruitment and polarization of macrophages in several cancers. We found that murine GL261 glioma cells overexpress GM‐CSF (granulocyte–macrophage colony‐stimulating factor encoded by the CSF2 gene) but not M‐CSF when compared to normal astrocytes. Knockdown of GM‐CSF in GL261 glioma cells strongly reduced microglia‐dependent invasion in organotypical brain slices and growth of intracranial gliomas and extended animal survival. The number of infiltrating microglia/macrophages (Iba1⁺ cells) and intratumoural angiogenesis were reduced in murine gliomas depleted of GM‐CSF. M1/M2 gene profiling in sorted microglia/macrophages suggests impairment of their pro‐invasive activation in GM‐CSF‐depleted gliomas. Deficiency of M‐CSF (op/op mice) did not affect glioma growth in vivo and the accumulation of Iba1⁺ cells, but impaired accumulation of Iba1⁺ cells in response to demyelination. These results suggest that distinct cytokines of the CSF family contribute to macrophage infiltration of tumours and in response to injury. The expression of CSF2 (but not CSF1) was highly up‐regulated in glioblastoma patients and we found an inverse correlation between CSF2 expression and patient survival. Therefore we propose that GM‐CSF triggers and drives the alternative activation of tumour‐infiltrating microglia/macrophages in which these cells support tumour growth and angiogenesis and shape the immune microenvironment of gliomas. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Nodal promotes the generation of M2‐like macrophages and downregulates the expression of IL‐12

Nodal, a member of the TGF‐β superfamily, is an embryonic morphogen that is upregulated in different types of tumors. Nodal increases the tumorigenesis by inducing angiogenesis and promoting metastasis. Importantly, Nodal inhibition suppresses the growth and invasion of tumor. Since tumor‐associated macrophages (TAMs) are the major infiltrating leukocytes in most cancers, we investigated whether Nodal is involved in the differentiation of TAMs. Our results revealed that Nodal inhibition in tumor microenvironment upregulated the production of IL‐12 in macrophages and reversed TAMs to classically activated macrophage phenotype. In contrast, treatment with recombinant Nodal (rNodal) decreased the expression of IL‐12 in murine macrophages. Furthermore, rNodal promoted macrophage polarization to an alternatively activated macrophage‐like/TAM phenotype and modulated its function. These results suggest that Nodal may play an important role in macrophage polarization and downregulation of IL‐12. The rescued antitumor function of TAMs via the inhibition of Nodal expression could be a new therapeutic strategy for cancer treatment.     

Human macrophages primed with angiogenic factors show dynamic plasticity, irrespective of extracellular matrix components

Macrophages are important in inflammation as well as in tissue repair processes. They can be activated by various stimuli and classified into two major groups: M1 (classically activated) or M2 (alternatively activated). Inflammation, angiogenesis and matrix remodeling play a major role in tissue repair. Here, we investigate the combined influence of a pro-angiogenic microenvironment and specific extracellular matrix (ECM) components or tissue culture polystyrene (TCPS) on the dynamics of human macrophage polarization. We established that human angiogenically primed macrophages cultured on different ECM components exhibit an M2-like polarization. These M2-like macrophages polarized to M1 and M2 macrophages with classical (LPS and IFNγ) stimuli and alternative (IL-4 and IL-13) stimuli respectively. Moreover, these M1 and M2 (primary) polarized macrophages rapidly underwent a secondary (re)polarization to M2 and M1 with conditioned media from M2 and M1 primary polarized macrophages respectively. In these initial priming and later (re)polarization processes the soluble factors had a dominant and orchestrating role, while the type of ECM (collagen I, fibronectin, versus tissue culture polystyrene) did not play a crucial role on the polarization of macrophages.     

IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion

Innate immune cells can constitute a substantial proportion of the cells within the tumor microenvironment and have been associated with tumor malignancy in patients and animal models of cancer; however, the mechanisms by which they modulate cancer progression are incompletely understood. Here, we show that high levels of cathepsin protease activity are induced in the majority of macrophages in the microenvironment of pancreatic islet cancers, mammary tumors, and lung metastases during malignant progression. We further show that tumor-associated macrophage (TAM)-supplied cathepsins B and S are critical for promoting pancreatic tumor growth, angiogenesis, and invasion in vivo, and markedly enhance the invasiveness of cancer cells in culture. Finally, we demonstrate that interleukin-4 (IL-4) is responsible for inducing cathepsin activity in macrophages in vitro and in vivo. Together, these data establish IL-4 as an important regulator, and cathepsin proteases as critical mediators, of the cancer-promoting functions of TAMs.     

Macrophage polarization in response to wear particles in vitro

Total joint replacement is a highly successful surgical procedure for treatment of patients with disabling arthritis and joint dysfunction. However, over time, with high levels of activity and usage of the joint, implant wear particles are generated from the articulating surfaces. These wear particles can lead to activation of an inflammatory reaction, and subsequent bone resorption around the implant （periprosthetic osteolysis）. Cells of the monocyte/macrophage lineage orchestrate this chronic inflammatory response, which is dominated by a pro-inflammatory （M 1） macrophage phenotype rather than an anti-inflammatory pro-tissue healing （M2） macrophage phenotype. While it has been shown that interleukin-4 （IL-4） selectively polarizes macrophages towards an M2 anti-inflammatory phenotype which promotes bone healing, rather than inflammation, little is known about the time course in which this occurs or conditions in which repolarization through I L-4 is most effective. The goal of this work was to study the time course of murine macrophage polarization and cytokine release in response to challenge with combinations of polymethyl methacrylate （PMMA） particles, lipopolysaccharide （LPS） and IL-4 in vitro. Treatment of particle-challenged monocyte/macrophages with IL-4 led to an initial suppression of pro-inflammatory cytokines and inducible nitric oxide synthase （iNOS） production and subsequent polarization into an M2 anti-inflammatory phenotype. This result was optimized when IL-4 was delivered before PMMA particle challenge, to an M 1 phenotype rather than to uncommitted （MO） macrophages. The effects of this polarization were sustained over a 5-day time course. Polarization of M1 macrophages into an M2 phenotype may be a strategy to mitigate wear particle associated periprosthetic osteolysis.     

Schistosoma japonicum infection induces macrophage polarization

The role of macrophages （MФ） as the first line of host defense is well accepted. These cells play a central role in orchestrating crucial functions during schistosomal infection. Thus, understanding the functional diversity of these cells in the process of infection as well as the mechanisms underlying these events is crucial for developing disease control strategies. In this study, we adopted a Mqb polarization recognition system. M1 macrophage was characterized by expressing CD16/32, IL-12 and iNOS. M2 macrophage was characterized by expressing CD206, IL-10 and arg-1. In vivo （mouse peritoneal macrophages of different infection stages were obtained） and in vitro （different S. japonicum antigens were used to stimulate RAW264.7） were characterized by using the above mentioned system. NCA and ACA stimulated RAW264.7 express significantly higher levels of IL-12 while significantly higher levels of IL-10 were detected after soluble egg antigen （SEA） stimulation. The results showed that dramatic changes of antigen in the microenvironment before and after egg production led to macrophage polarization. Furthermore, through TLR blocking experiments, the TLR4 signaling pathway was found to play a role in the process of macrophage polarization toward M1. Our data suggest that macrophage polarization during S. japonicum infection had significant effects on host immune responses to S. japonicum.     

Critical role of p38 MAPK in IL‐4‐induced alternative activation of peritoneal macrophages

Alternative activation of macrophages plays an important role in a range of physiological and pathological processes. This alternative phenotype, also known as M2 macrophages, is induced by type 2 cytokines such as IL‐4. The binding of IL‐4 to its receptor leads to activation of two major signaling pathways: STAT‐6 and PI3K. However, recent studies have described that p38 MAPK might play a role in IL‐4‐dependent signaling in some cells, although its role in macrophages is still controversial. In this study, we investigated whether p38 MAPK plays a role in the polarization of macrophages in mice. Our results reveal that IL‐4 induces phosphorylation of p38 MAPK in thioglycollate‐elicited murine peritoneal macrophages, in addition to STAT‐6 and PI3K activation. Furthermore, p38 MAPK inactivation, by gene silencing or pharmacological inhibition, suppressed IL‐4‐induced typical M2 markers, indicating the involvement of p38 MAPK in the signaling of IL‐4 leading to M2‐macrophage polarization. Moreover, p38 MAPK inhibition blocked phosphorylation of STAT‐6 and Akt, suggesting that p38 MAPK is upstream of these signaling pathways. Finally, we show that in an in vivo model of chitin‐induced M2 polarization, p38 MAPK inhibition also diminished activation of M2 markers. Taken together, our data establish a new role for p38 MAPK during IL‐4‐induced alternative activation of macrophages.     

Transient depletion of CD4+ CD25+ regulatory T cells results in multiple autoimmune diseases in wild‐type and B‐cell‐deficient NOD mice

Plasticity is a hallmark of macrophages, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic (M1) and alternative (M2). Rapamycin (RAPA) is crucial for survival and functions of myeloid phagocytes, but its effects on macrophage polarization are not yet studied. To address this issue, human macrophages obtained from six normal blood donors were polarized to M1 or M2 in vitro by lipopolysaccharide plus interferon-γ or interleukin-4 (IL-4), respectively. The presence of RAPA (10 ng/ml) induced macrophage apoptosis in M2 but not in M1. Beyond the impact on survival in M2, RAPA reduced CXCR4, CD206 and CD209 expression and stem cell growth factor-β, CCL18 and CCL13 release. In contrast, in M1 RAPA increased CD86 and CCR7 expression and IL-6, tumour necrosis factor-α and IL-1β release but reduced CD206 and CD209 expression and IL-10, vascular endothelial growth factor and CCL18 release. In view of the in vitro data, we examined the in vivo effect of RAPA monotherapy (0·1 mg/kg/day) in 12 patients who were treated for at least 1 month before islet transplant. Cytokine release by Toll-like receptor 4-stimulated peripheral blood mononuclear cells showed a clear shift to an M1-like profile. Moreover, macrophage polarization 21 days after treatment showed a significant quantitative shift to M1. These results suggest a role of mammalian target of rapamycin (mTOR) into the molecular mechanisms of macrophage polarization and propose new therapeutic strategies for human M2-related diseases through mTOR inhibitor treatment.     

Normal and SV40 transfected human peritoneal mesothelial cells produce IL-6 and IL-8: implication for gynaecological disease

High levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) have been demonstrated in the peritoneal fluid of benign and malignant gynaecological disease. Peritoneal monocytes and macrophages, endometrial cells, endometrial and peritoneal stromal cells and tumour cells produce these cytokines in vitro. To investigate whether normal human peritoneal mesothelial cells (HPMC) produce IL-6 and IL-8, HPMC were isolated from omental biopsies. Primary HPMC (P-HPMC) were transfected with pSV3-neo encoding SV40 large T antigen (T-HPMC) to generate sufficient cells. T-HPMC preserved the characteristics of P-HPMC as assessed by phase contrast microscopy, electron microscopy, immunocytochemistry and flow cytometry (FACS) analysis. T-HPMC retained a stable phenotype up to passage 14-19, whereas P-HPMC proliferated poorly and became senescent by passage 4-6. T-HPMC and P-HPMC constitutively expressed IL-6 and IL-8 at both protein and mRNA level. IL-6 and IL-8 production was stimulated by recombinant human interleukin-1beta (hIL-1beta) or human tumour necrosis factor-alpha (hTNF-alpha) alone in a dose-dependent manner. Moreover, hIL-1beta or hTNF-alpha up-regulated IL-6 and IL-8 gene expression as determined by competitive PCR. In contrast, human interferon-gamma (hIFN-gamma) or lipopolysaccharide (LPS) showed no effect. These data indicate that (1) T-HPMC lines mimic the morphological and functional features of P-HPMC, (2) P-HPMC and T-HPMC constituitively produce IL-6 and IL-8, which is enhanced by hIL-1beta and hTNF-alpha and (3) HPMC in vivo may participate in the pathogenesis of benign and malignant gynaecological disease.     

IL-10 regulation of macrophage VEGF production is dependent on macrophage polarisation and hypoxia

Vascular endothelial growth factor A (VEGF) is critical for vascular remodelling during tissue repair subsequent to inflammation or injury, but under pathological conditions, VEGF induces tissue damaging angiogenesis. Macrophages generate VEGF that supports angiogenesis, when they adapt to their environment and respond with a co-ordinated set of signals to promote or resolve inflammation. Depending on the stimulus, the phenotype of macrophage activation is broadly classified into M1 (NOS2⁺) and M2 (arginase-1⁺). In recent studies, IL-10, an anti-inflammatory cytokine that suppresses the M1 phenotype, has been shown to dampen the angiogenic switch and subsequent neovascularisation. However, as we show here, these effects are context dependent. In this study, we have demonstrated that IL-10 inhibits M1 bone marrow-derived macrophages (BMDMs) VEGF, stimulated by LPS/CGS21680 (adenosine A2A receptor agonist), but does not prevent VEGF production from M2 macrophages stimulated with prostaglandin E2 (PGE2). Furthermore, we show that hypoxic-conditioned BMDM generated VEGF was maintained in the presence of IL-10, but was suppressed when concomitantly stimulated with IFN-γ. Finally, LPS/PGE2 generated an arginase-1⁺ M2 macrophage that in addition to generating VEGF produced significant quantities of IL-10. Under these conditions, neither in IL-10 deficient macrophages nor following IL-10 neutralization was VEGF production affected. Our results indicate IL-10 suppressed M1 but not M2 derived VEGF, and that activation signals determined the influence of IL-10 on VEGF production. Consequently, therapies to suppress macrophage activation that as a result generate IL-10, or utilising IL-10 as a potential anti-angiogenic therapy, may result in a paradoxical support of neovascularisation and thus on-going tissue damage or aberrant repair.     

Alternative Reprogramming of M1/M2 Phenotype of Mouse Peritoneal Macrophages In Vitro with Interferon-γ and Interleukin-4

An important role in the development of the immune response is played by macrophages that acquire either anti-inflammatory M1 or anti-inflammatory M2 phenotype depending on their microenvironment. The possibility of targeted reprogramming of the initial M2 macrophage phenotype towards M1 phenotype and vice versa using macrophage reprogramming factors IFN-γ and IL-4, respectively, was demonstrated. We showed that macrophages of genetically different mouse strains did not practically differ by their reprogramming capacity. Our findings suggest that macrophage programming not only participates in the triggering of the immune response, but also can ensure plasticity of functional activity during the developing response.     

Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation

The aim of this study was to investigate the mechanisms by which interleukin-10 (IL-10) induces tumour growth in a mouse-melanoma model. A B16-melanoma cell line (B16-0) was transfected with IL-10 cDNA and three clones that secreted high (B16-10), medium and low amounts of IL-10 were selected. Cell proliferation and IL-10 production were compared in vitro, and tumour growth, percentages of necrotic areas, tumour cells positive for proliferating cell nuclear antigen (PCNA), IL-10 receptor (IL-10R) and major histocompatibility complex type I (MHC-I) and II (MHC-II), as well as infiltration of macrophages, CD4+ and CD8+ lymphocytes and blood vessels were compared in vivo among IL-10-transfected and non-transfected tumours. Proliferation and tumour growth were greater for IL-10-transfected than for non-transfected cells (P < 0.001), and correlated with IL-10 concentration (r > or =0.79, P < 0.006). Percentages of tumour cells positive for PCNA and IL-10R were 4.4- and 16.7-fold higher, respectively, in B16-10 than in B16-0 tumours (P < 0.001). Macrophage distribution changed from a diffuse pattern in non-transfected (6.4 +/- 1.7%) to a peripheral pattern in IL-10-transfected (3.8 +/- 1.7%) tumours. The percentage of CD4+ lymphocytes was 7.6 times higher in B16-10 than in B16-0 tumours (P = 0.002). The expression of MHC-I molecules was present in all B16-0 tumour cells and completely negative in B16-10 tumour cells. In B16-0 tumours, 89 +/- 4% of the whole tumour area was necrotic, whereas tumours produced by B16-10 cells showed only 4.3 +/- 6% of necrotic areas. IL-10-transfected tumours had 17-fold more blood vessels than non-transfected tumours (61.8 +/- 8% versus 3.5 +/- 1.7% blood vessels/tumour; P < 0.001). All the effects induced by IL-10 were prevented in mice treated with a neutralizing anti-IL-10 monoclonal antibody. These data indicate that IL-10 could induce tumour growth in this B16-melanoma model by stimulation of tumour-cell proliferation, angiogenesis and immunosuppression.     

Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds

In normal tissue repair, macrophages exhibit a pro-inflammatory phenotype (M1) at early stages and a pro-healing phenotype (M2) at later stages. We have previously shown that M1 macrophages initiate angiogenesis while M2 macrophages promote vessel maturation. Therefore, we reasoned that scaffolds that promote sequential M1 and M2 polarization of infiltrating macrophages should result in enhanced angiogenesis and healing. To this end, we first analyzed the in vitro kinetics of macrophage phenotype switch using flow cytometry, gene expression, and cytokine secretion analysis. Then, we designed scaffolds for bone regeneration based on modifications of decellularized bone for a short release of interferon-gamma (IFNg) to promote the M1 phenotype, followed by a more sustained release of interleukin-4 (IL4) to promote the M2 phenotype. To achieve this sequential release profile, IFNg was physically adsorbed onto the scaffolds, while IL4 was attached via biotin-streptavidin binding. Interestingly, despite the strong interactions between biotin and streptavidin, release studies showed that biotinylated IL4 was released over 6 days. These scaffolds promoted sequential M1 and M2 polarization of primary human macrophages as measured by gene expression of ten M1 and M2 markers and secretion of four cytokines, although the overlapping phases of IFNg and IL4 release tempered polarization to some extent. Murine subcutaneous implantation model showed increased vascularization in scaffolds releasing IFNg compared to controls. This study demonstrates that scaffolds for tissue engineering can be designed to harness the angiogenic behavior of host macrophages towards scaffold vascularization.     

Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype

M1 macrophages play a major role in worsening muscle injury in the mdx mouse model of Duchenne muscular dystrophy. However, mdx muscle also contains M2c macrophages that can promote tissue repair, indicating that factors regulating the balance between M1 and M2c phenotypes could influence the severity of the disease. Because interleukin-10 (IL-10) modulates macrophage activation in vitro and its expression is elevated in mdx muscles, we tested whether IL-10 influenced the macrophage phenotype in mdx muscle and whether changes in IL-10 expression affected the pathology of muscular dystrophy. Ablation of IL-10 expression in mdx mice increased muscle damage in vivo and reduced mouse strength. Treating mdx muscle macrophages with IL-10 reduced activation of the M1 phenotype, assessed by iNOS expression, and macrophages from IL-10 null mutant mice were more cytolytic than macrophages isolated from wild-type mice. Our data also showed that muscle cells in mdx muscle expressed the IL-10 receptor, suggesting that IL-10 could have direct effects on muscle cells. We assayed whether ablation of IL-10 in mdx mice affected satellite cell numbers, using Pax7 expression as an index, but found no effect. However, IL-10 mutation significantly increased myogenin expression in vivo during the acute and the regenerative phase of mdx pathology. Together, the results show that IL-10 plays a significant regulatory role in muscular dystrophy that may be caused by reducing M1 macrophage activation and cytotoxicity, increasing M2c macrophage activation and modulating muscle differentiation.     

Pro-tumour activity of interleukin-22 in HPAFII human pancreatic cancer cells

Interleukin (IL)-22 is a cytokine involved in inflammatory and wound healing processes that is secreted primarily by T helper type 17 (Th17) cells. IL-22 receptor (IL-22R) expression is limited to epithelial cells of the digestive organs, respiratory tract and skin. Most tumours originating in these sites over-express IL-22R. Interestingly, there is an increase in Th17 frequency within the peripheral blood and tumour microenvironment of advanced cancer patients. Subsequently, IL-17 has been shown to display both pro-tumour and anti-tumour functions. Because many tumours lack expression of the IL-17 receptor, the effects of IL-17 on tumour growth are generated by cells that surround the tumour cells. Like IL-17, high levels of IL-22 have been detected in tumour tissues and the peripheral blood of cancer patients; however, the direct effect of IL-22 on tumour cells has remained largely unknown. In this report, we show that IL-22 stimulated production of vascular endothelial growth factor (VEGF) and the anti-apoptotic factor Bcl-X(L) in IL-22R-positive HPAFII human pancreatic cancer cells. Additionally, IL-22 augmented HPAFII cell production of immunosuppressive cytokines. We show further that IL-22 activation of HPAFII cells diminished T cell production of interferon (IFN)-γ through the action of IL-10. Strikingly, we show for the first time that IL-22 can fully protect cancer cells from natural killer (NK) cell-mediated cytotoxicity by stimulating tumour production of IL-10 and transforming growth factor (TGF)-β1. Our data support the idea that IL-22 may act to promote the pathogenesis of cancers rather than function in anti-tumour immunity.