Nicholas J. Wilson

IL-12 and IL-23: master regulators of innate and adaptive immunity.

Summary:  Initiation of an effective immune response requires close interactions between innate and adaptive immunity. Recent advances in the field of cytokine biology have led to an increased understanding of how myeloid cell‐derived factors regulate the immune system to protect the host from infections and prevent tumor development. In this review, we focus on the function of interleukin (IL)‐23, a new member of the IL‐12 family of regulatory cytokines produced by activated macrophages and dendritic cells. We propose that IL‐12 and IL‐23 promote two distinct immunological pathways that have separate but complementary functions. IL‐12 is required for antimicrobial responses to intracellular pathogens, whereas IL‐23 is likely to be important for the recruitment and activation of a range of inflammatory cells that is required for the induction of chronic inflammation and granuloma formation. These two cytokines work in concert to regulate cellular immune responses critical for host defense and tumor suppression.

Roles of the Mitogen-activated Protein Kinase Family in Macrophage Responses to Colony Stimulating Factor-1 Addition and Withdrawal

Colony stimulating factor-1 (CSF-1) (or macrophage CSF) is involved in the survival, proliferation, differentiation, and activation of cells of the monocyte/macrophage lineage. Because the mitogen-activated protein kinase family members extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinase are widely implicated in such cellular functions, we measured their activity in growing and growth-arrested cultures of bone marrow-derived macrophages (BMM), as well as their stimulation by saturating concentrations of CSF-1. ERK activity was approximately 2-fold higher in cycling BMM compared with growth-arrested BMM; in addition, CSF-1-stimulated BMM DNA synthesis was partially inhibited by PD98059, a specific inhibitor of MEK activation, suggesting a role for a mitogen-activated protein-ERK kinase (MEK)/ERK pathway in the control of DNA synthesis but surprisingly not in the control of cyclin D1 mRNA or c-myc mRNA expression. The suppression of BMM apoptosis by CSF-1, i.e. enhanced survival, was not reversed by PD98059, suggesting that a MEK/ERK pathway is not involved in this process. Using a quantitative kinase assay, it was found that CSF-1 gave a slight increase in BMM p38 activity, supporting prior data that CSF-1 is a relatively weak stimulator of inflammatory cytokine production in monocytes/macrophages. Relatively high concentrations of the p38 inhibitor, SKB202190, suppressed CSF-1-stimulated BMM DNA synthesis. No evidence could be obtained for the involvement of p38 activity in BMM apoptosis following CSF-1 withdrawal. We were not able to show that CSF-1 enhanced BMM JNK-1 activity to a significant extent; again, no role could be found for JNK-1 activity in the BMM apoptosis occurring after CSF-1 removal.

Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells.

Using two‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2‐D SDS‐PAGE) of 32P‐labeled cytosolic and membrane extracts, we identified a 21.5 kDa phosphoprotein with an isoelectric point of 6.0 in NFS‐60 cells that was phosphorylated maximally at 15 min by treatment with granulocyte‐colony stimulating factor (G‐CSF) but not with interlevkin‐3 (IL‐3) or colony‐stimulating factor‐1 (macrophage‐colony stimulating factor (CSF‐1 (M‐CSF)). The phosphorylation of this protein, designated 21.5/6.0, was unaffected by a series of antiproliferative agents [32]. These findings suggested that the 21.5/6.0 phosphoprotein may be involved in specific G‐CSF‐mediated biological responses such as activation and/or differentiation. We sought to characterize this 21.5/6.0 by a novel combination of 2‐D SDS‐PAGE and hydroxyapatite (HTP)‐chromatography. Amino acid sequence determination of 21.5/6.0 revealed it to share a high level of homology with copper/zinc superoxide dismutase (Cu/Zn‐SOD), indicating that a Cu/Zn‐SOD is phosphorylated following treatment with G‐CSF. This is the first report of the phosphorylation and possible involvement of Cu/Zn‐SOD protein in granulocyte activation/differentiation events. In addition, Cu/Zn‐SOD levels and activity were diminished by G‐CSF but not IL‐3 treatment. This new protocol combining 2‐D SDS‐PAGE and HTP‐chromatography allows the characterization of low abundance phosphoproteins involved in the cellular responses to G‐CSF and presumably to other cytokines/growth factors.

Short-chain fatty acids reduce expression of specific protein kinase C isoforms in human colonic epithelial cells

LIM1215 colon cancer cells were used as a model of human colonic epithelium to examine the effects of butyrate on protein kinase C (PKC) activity and isoform expression. On Western blot analysis, LIM1215 cells express the PKC isoforms α, β, ε, ζ, and λ, but not γ, θ, or μ. Treatment with 2 mM butyrate for 48 h reduced cellular PKC activity up to 50% and specifically reduced the expression of PKCα and PKCε. Similar results were obtained using Caco‐2 colon cancer cells. These effects were neither a consequence of the induction of differentiation itself nor the result of direct or indirect activation of PKC. Although dependent on gene transcription and protein synthesis, the effect was not due to a reduction in the synthesis of PKC protein. Butyrates effect was independent of its β‐oxidation but was mimicked, at least in part, by trichostatin A, an inhibitor of histone deacetylase. J. Cell. Physiol. 182:222–231, 2000.

Colony-stimulating factor-1 (CSF-1) receptor-mediated macrophage differentiation in myeloid cells: a role for tyrosine 559-dependent protein phosphatase 2A (PP2A) activity.

M1 myeloid cells transfected with the wild-type (WT) colony-stimulating factor-1 (CSF-1) receptor (CSF-1R; M1/WT cells) undergo CSF-1-dependent macrophage differentiation. By mutation studies, we have provided prior evidence that tyrosine 559 in the CSF-1R cytoplasmic domain governs the Src-dependent differentiation pathway. Further components of this pathway were then sought. We report that the extent of CSF-1-mediated tyrosine phosphorylation of protein phosphatase 2A (PP2A), and the associated loss of its activity were reduced in M1 cells transfected with the CSF-1R with a tyrosine-to-phenylalanine mutation at position 559 (M1/559 cells), compared with the corresponding responses in CSF-1-treated M1/WT cells. This evidence for an involvement of a reduction in PP2A activity in the differentiation process was supported by the restoration of the defect in the CSF-1-mediated differentiation of M1/559 cells by the addition of the PP2A inhibitor, okadaic acid. It was also found that the degree of activation of extracellular-signal-regulated kinase (ERK) activities by CSF-1 was reduced in M1/559 cells, suggesting their involvement in the differentiation process. These data suggest that PP2A and ERK form part of the Src-dependent signal-transduction cascade governing CSF-1-mediated macrophage differentiation in M1 cells.

A novel 110 kDa form of myosin XVIIIA (MysPDZ) is tyrosine-phosphorylated after colony-stimulating factor-1 receptor signalling.

Macrophage colony-stimulating factor (M-CSF or CSF-1) controls the development of macrophage lineage cells via activation of its tyrosine kinase receptor, c-Fms. After adding CSF-1 to M1 myeloid cells expressing CSF-1R (CSF-1 receptor), tyrosine phosphorylation of many cellular proteins occurs, which might be linked to subsequent macrophage differentiation. The biological significance and characterization of such proteins were explored by a dual strategy comprising two-dimensional SDS/PAGE analysis of cell lysates of CSF-1-treated M1 cells expressing the wild-type or a mutated receptor, together with an enrichment strategy involving a tyrosine-phosphorylated receptor construct. In the present study, we report the identification by MS of a novel, low-abundance, 110 kDa form of myosin XVIIIA (MysPDZ, myosin containing PDZ domain), which appears to be preferentially tyrosine-phosphorylated after CSF-1R activation when compared with other known isoforms. Receptor mutation studies indicate that CSF-1R-dependent tyrosine phosphorylation of p110myosin XVIIIA requires Tyr-559 in the cytoplasmic domain of the receptor and is therefore Src-family kinase-dependent. Gelsolin, Erp61 protein disulphide-isomerase and possibly non-muscle myosin IIA were also tyrosine-phosphorylated under similar conditions. Similar to the more abundant p190 isoform, p110 myosin XVIIIA lacks a PDZ domain and, in addition, it may lack motor activity. The phosphorylation of p110 myosin XVIIIA by CSF-1 may alter its cellular localization or target its association with other proteins.

cAMP inhibits CSF‐1‐stimulated tyrosine phosphorylation but augments CSF‐1R‐mediated macrophage differentiation and ERK activation

Macrophage colony stimulating factor (M‐CSF) or CSF‐1 controls the development of the macrophage lineage through its receptor tyrosine kinase, c‐Fms. cAMP has been shown to influence proliferation and differentiation in many cell types, including macrophages. In addition, modulation of cellular ERK activity often occurs when cAMP levels are raised. We have shown previously that agents that increase cellular cAMP inhibited CSF‐1‐dependent proliferation in murine bone marrow‐derived macrophages (BMM) which was associated with an enhanced extracellular signal‐regulated kinase (ERK) activity. We report here that increasing cAMP levels, by addition of either 8‐bromo cAMP (8BrcAMP) or prostaglandin E1 (PGE1), can induce macrophage differentiation in M1 myeloid cells engineered to express the CSF‐1 receptor (M1/WT cells) and can potentiate CSF‐1‐induced differentiation in the same cells. The enhanced CSF‐1‐dependent differentiation induced by raising cAMP levels correlated with enhanced ERK activity. Thus, elevated cAMP can promote either CSF‐1‐induced differentiation or inhibit CSF‐1‐induced proliferation depending on the cellular context. The mitogen‐activated protein kinase/extracellular signal‐related protein kinase kinase (MEK) inhibitor, PD98059, inhibited both the cAMP‐ and the CSF‐1R‐dependent macrophage differentiation of M1/WT cells suggesting that ERK activity might be important for differentiation in the M1/WT cells. Surprisingly, addition of 8BrcAMP or PGE1 to either CSF‐1‐treated M1/WT or BMM cells suppressed the CSF‐1R‐dependent tyrosine phosphorylation of cellular substrates, including that of the CSF‐1R itself. It appears that there are at least two CSF‐1‐dependent pathway(s), one MEK/ERK dependent pathway and another controlling the bulk of the tyrosine phosphorylation, and that cAMP can modulate signalling through both of these pathways.

Therapeutic Targeting of the G-CSF Receptor Reduces Neutrophil Trafficking and Joint Inflammation in Antibody-Mediated Inflammatory Arthritis

G-CSF is a hemopoietic growth factor that has a role in steady state granulopoiesis, as well as in mature neutrophil activation and function. G-CSF– and G-CSF receptor–deficient mice are profoundly protected in several models of rheumatoid arthritis, and Ab blockade of G-CSF also protects against disease. To further investigate the actions of blocking G-CSF/G-CSF receptor signaling in inflammatory disease, and as a prelude to human studies of the same approach, we developed a neutralizing mAb to the murine G-CSF receptor, which potently antagonizes binding of murine G-CSF and thereby inhibits STAT3 phosphorylation and G-CSF receptor signaling. Anti–G-CSF receptor rapidly halted the progression of established disease in collagen Ab-induced arthritis in mice. Neutrophil accumulation in joints was inhibited, without rendering animals neutropenic, suggesting an effect of G-CSF receptor blockade on neutrophil homing to inflammatory sites. Consistent with this, neutrophils in the blood and arthritic joints of anti–G-CSF receptor–treated mice showed alterations in cell adhesion receptors, with reduced CXCR2 and increased CD62L expression. Furthermore, blocking neutrophil trafficking with anti–G-CSF receptor suppressed local production of proinflammatory cytokines (IL-1β, IL-6) and chemokines (KC, MCP-1) known to drive tissue damage. Differential gene expression analysis of joint neutrophils showed a switch away from an inflammatory phenotype following anti–G-CSF receptor therapy in collagen Ab-induced arthritis. Importantly, G-CSF receptor blockade did not adversely affect viral clearance during influenza infection in mice. To our knowledge, we describe for the first time the effect of G-CSF receptor blockade in a therapeutic model of inflammatory joint disease and provide support for pursuing this therapeutic approach in treating neutrophil-associated inflammatory diseases.

Production of a human neutralizing monoclonal antibody and its crystal structure in complex with ectodomain 3 of the interleukin-13 receptor α1.

Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.

The Critical Role of the Colony-Stimulating Factor-1 Receptor in the Differentiation of Myeloblastic Leukemia Cells

How diverse stimuli control hemopoietic lineage development is unknown. An early event during induction of macrophage differentiation in the myeloblastic leukemia M1 cell line by different stimuli, such as leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), is expression of the colony-stimulating factor-1 receptor (CSF-1R). We report that expression of active CSF-1R in M1 cells accelerated their subsequent terminal differentiation into macrophages in response to LIF and IL-6 when compared with cells lacking the CSF-1R or expressing the receptor with compromised kinase activity; however, there was no requirement for signaling through the CSF-1R, for example, via endogenous CSF-1, during the actual LIF-induced and IL-6–induced differentiation stage. Differences were noted in the signaling pathways downstream of the LIF receptor depending on the presence of the CSF-1R. Both LIF and IL-6 gave an additive response with CSF-1, consistent with LIF and IL-6 acting via a different signaling pathway (signal transducer and activator of transcription 3 dependent) than CSF-1 (extracellular signal-regulated kinase dependent). Based at least on this cell model, we propose that terminal macrophage differentiation involves a critical priming or deterministic phase in which signaling by the CSF-1R prepares a precursor population for subsequent rapid terminal macrophage differentiation by diverse stimuli. We also propose that expression and activation of the CSF-1R explain much prior literature on macrophage lineage commitment in M1 leukemic cells and may be important in controlling the progression of certain myeloid leukemias. (Mol Cancer Res 2008;6(3):458–67)