C5a initiates the inflammatory cascade in immune complex peritonitis

Immune complex (IC)-induced inflammation is integral to the pathogenesis of several autoimmune diseases. ICs activate the complement system and interact with IgG FcgammaR. In this study, we demonstrate that activation of the complement system, specifically generation of C5a, initiates the neutrophilic inflammation in IC peritonitis. We show that ablation of C5a receptor signaling abrogates neutrophil recruitment in wild-type mice and prevents the enhancement of neutrophil migration seen in FcgammaRIIB(-/-) mice, suggesting that C5aR signaling is the crucial initial event upstream of FcgammaR signaling. We also provide evidence that C5a initiates the inflammatory cascade both directly, through C5aR-mediated effector functions on infiltrating and resident peritoneal cells, and indirectly, through shifting the balance between activating and inhibitory FcgammaRs on resident cells toward an inflammatory phenotype. We conclude that complement activation and C5a generation are prerequisites for IC-induced inflammation through activating FcgammaR, which amplifies complement-induced inflammation in autoimmunity.     

Tissue- and stimulus-dependent role of phosphatidylinositol 3-kinase isoforms for neutrophil recruitment induced by chemoattractants in vivo

PI3K plays a fundamental role in regulating neutrophil recruitment into sites of inflammation but the role of the different isoforms of PI3K remains unclear. In this study, we evaluated the role of PI3Kgamma and PI3Kdelta for neutrophil influx induced by the exogenous administration or the endogenous generation of the chemokine CXCL1. Administration of CXCL1 in PI3Kgamma(-/-) or wild-type (WT) mice induced similar increases in leukocyte rolling, adhesion, and emigration in the cremaster muscle when examined by intravital microscopy. The induction of neutrophil recruitment into the pleural cavity or the tibia-femoral joint induced by the injection of CXCL1 was not significantly different in PI3Kgamma(-/-) or WT mice. Neutrophil influx was not altered by treatment of WT mice with a specific PI3Kdelta inhibitor, IC87114, or a specific PI3Kgamma inhibitor, AS605240. The administration of IC87114 prevented CXCL1-induced neutrophil recruitment only in presence of the PI3Kgamma inhibitor or in PI3Kgamma(-/-) mice. Ag challenge of immunized mice induced CXCR2-dependent neutrophil recruitment that was inhibited by wortmannin or by blockade of and PI3Kdelta in PI3Kgamma(-/-) mice. Neutrophil recruitment to bronchoalveolar lavage induced by exogenously added or endogenous production of CXCL1 was prevented in PI3Kgamma(-/-) mice. The accumulation of the neutrophils in lung tissues was significantly inhibited only in PI3Kgamma(-/-) mice treated with IC87114. Neutrophil recruitment induced by exogenous administration of C5a or fMLP appeared to rely solely on PI3Kgamma. Altogether, our data demonstrate that there is a tissue- and stimulus-dependent role of PI3Kgamma and PI3Kdelta for neutrophil recruitment induced by different chemoattractants in vivo.     

The urokinase/urokinase receptor system mediates the IgG immune complex-induced inflammation in lung

Immune complex (IC) deposition induces an acute inflammatory response with tissue injury. IC-induced inflammation is mediated by inflammatory cell infiltration, a process highly regulated by the cell surface-specific receptor (uPAR), a binding partner for the urokinase-type plasminogen activator (uPA). We assessed the role of the uPA/uPAR system in IC-induced inflammation using the pulmonary reverse passive Arthus reaction in mice lacking uPA and uPAR compared with their corresponding wild-type controls. Both uPA-deficient C57BL/6J (uPA(-/-)) and uPAR-deficient mice on a mixed C57BL/6J (75%) x 129 (25%) background (uPAR(-/-)) demonstrated a marked reduction of the inflammatory response due to decreased production of proinflammatory mediators TNF-alpha and Glu-Leu-Arg (ELR)-CXC chemokine MIP-2. In uPAR(-/-) animals, the reduction of inflammatory response was more pronounced because of decreased migratory capacity of polymorphonuclear leukocytes. We show that the uPA/uPAR system is activated in lung of wild-type mice, particularly in resident alveolar macrophages (AM), early in IC-induced alveolitis. This activation is necessary for an adequate C5a anaphylatoxin receptor signaling on AM that, in turn, modulates the functional balance of the activating/inhibitory IgG FcgammaRs responsible for proinflammatory mediator release. These data provide the first evidence that the uPA/uPAR plays an important immunoregulatory role in the initiation of the reverse passive Arthus reaction in the lung by setting the threshold for C5a anaphylatoxin receptor/FcgammaR activation on AM. The findings indicate an important link between the uPA/uPAR system and the two main components involved in the IC inflammation, namely, complement and FcgammaRs.     

Suppressors of cytokine signaling regulate Fc receptor signaling and cell activation during immune renal injury

Suppressors of cytokine signaling (SOCS) are cytokine-inducible proteins that modulate receptor signaling via tyrosine kinase pathways. We investigate the role of SOCS in renal disease, analyzing whether SOCS regulate IgG receptor (FcgammaR) signal pathways. In experimental models of immune complex (IC) glomerulonephritis, the renal expression of SOCS family genes, mainly SOCS-3, significantly increased, in parallel with proteinuria and renal lesions, and the proteins were localized in glomeruli and tubulointerstitium. Induction of nephritis in mice with a deficiency in the FcgammaR gamma-chain (gamma(-/-) mice) resulted in a decrease in the renal expression of SOCS-3 and SOCS-1. Moreover, blockade of FcgammaR by Fc fragment administration in rats with ongoing nephritis selectively inhibited SOCS-3 and SOCS-1, without affecting cytokine-inducible Src homology 2-containing protein and SOCS-2. In cultured human mesangial cells (MC) and monocytes, IC caused a rapid and transient induction of SOCS-3 expression. Similar kinetics was observed for SOCS-1, whereas SOCS-2 expression was very low. MC from gamma(-/-) mice failed to respond to IC activation, confirming the participation of FcgammaR. Interestingly, IC induced tyrosine phosphorylation of SOCS-3 and Tec tyrosine kinase, and both proteins coprecipitated in lysates from IC-stimulated MC, suggesting intracellular association. IC also activated STAT pathway in MC, which was suppressed by SOCS overexpression, mainly SOCS-3. In SOCS-3 knockdown studies, specific antisense oligonucleotides inhibited mesangial SOCS-3 expression, leading to an increase in the IC-induced STAT activation. Our results indicate that SOCS may play a regulatory role in FcgammaR signaling, and implicate SOCS as important modulators of cell activation during renal inflammation.     

Macrophages induce the inflammatory response in the pulmonary Arthus reaction through G alpha i2 activation that controls C5aR and Fc receptor cooperation

Complement and FcgammaR effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory FcgammaRIIB, increased levels of activating FcgammaRIII, and highly induced FcgammaR-mediated TNF-alpha and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of G(i)-type G protein signaling in C5aR-mediated control of the regulatory FcgammaR system in vitro, and analysis of the various C5aR-, FcgammaR-, and G(i)-deficient mice verifies the importance of Galpha(i2)-associated C5aR and the FcgammaRIII-FcgammaRIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and FcgammaRIII-positive cells into C5aR- and FcgammaRIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or FcgammaRIII do not possess any such inducibility of immune complex disease, whereas reconstitution with FcgammaRIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a Galpha(i2)-dependent signal for modulating the two opposing FcgammaR, FcgammaRIIB and FcgammaRIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.     

A codominant role of Fc gamma RI/III and C5aR in the reverse Arthus reaction

Recent attempts to specify the relative contribution of FcR and complement in various experimental systems of immune complex disease have led to opposing conclusions. As concluded in IgG FcRgamma-/- mice, manifestation of disease is almost exclusively determined by FcgammaR on effector cells, arguing for a minor role of complement. In contrast, data obtained with C5aR-/- mice suggested that, dependent on the tissue site, complement is more important than FcgammaR. In this paper, we demonstrate that, in response to IgG immune complex formation, FcgammaRI/III- and C5aR-mediated pathways are both necessary and only together are they sufficient to trigger the full expression of inflammation in skin and lung. Moreover, both effector systems are not entirely independent, suggesting an interaction between FcgammaR and C5aR. Therefore, FcgammaR-mediated responses can be integrated through C5aR activation, which may explain why these two receptor pathways have previously been considered to dominate each other.     

Distinct tissue site-specific requirements of mast cells and complement components C3/C5a receptor in IgG immune complex-induced injury of skin and lung.

We induced the passive reverse Arthus reaction to IgG immune complexes (IC) at different tissue sites in mice lacking C3 treated or not with a C5aR-specific antagonist, or in mice lacking mast cells (Kit(W)/Kit(W-v) mice), and compared the inflammatory responses with those in the corresponding wild-type mice. We confirmed that IC inflammation of skin can be mediated largely by mast cells expressing C5aR and FcgammaRIII. In addition, we provided evidence for C3-independent C5aR triggering, which may explain why the cutaneous Arthus reaction develops normally in C3(-/-) mice. Furthermore, some, but not all, of the acute changes associated with the Arthus response in the lung were significantly more intense in normal mice than in C3(-/-) or Kit(W)/Kit(W-v) mice, indicating for C3- and mast cell-dependent and -independent components. Finally, we demonstrated that C3 contributed to the elicitation of neutrophils to alveoli, which corresponded to an increased synthesis of TNF-alpha, macrophage-inflammatory protein-2, and cytokine-induced neutrophil chemoattractant. While mast cells similarly influenced alveolar polymorphonuclear leukocyte influx, the levels of these cytokines remained largely unaffected in mast cell deficiency. Together, the phenotypes of C3(-/-) mice and Kit(W)/Kit(W-v) mice suggest that complement and mast cells have distinct tissue site-specific requirements acting by apparently distinct mechanisms in the initiation of IC inflammation.     

Sphingosine kinase 1 mediation of expression of the anaphylatoxin receptor C5L2 dampens the inflammatory response to endotoxin

The complement anaphylatoxin C5a has a pathogenetic role in endotoxin-induced lung inflammatory injury by regulating phagocytic cell migration and activation. Endotoxin and C5a activate the enzyme sphingosine kinase (Sphk) 1 to generate the signaling lipid sphingosine-1-phosphate (S1P), a critical regulator of phagocyte function. We assessed the function of Sphk1 and S1P in experimental lung inflammatory injury and determined their roles in anaphylatoxin receptor signaling and on the expression of the two C5a receptors, C5aR (CD88) and C5L2, on phagocytes. We report that Sphk1 gene deficient (Sphk1(-/-)) mice had augmented lung inflammatory response to endotoxin compared to wild type mice. Sphk1 was required for C5a-mediated reduction in cytokine and chemokine production by macrophages. Moreover, neutrophils from Sphk1(-/-) mice failed to upregulate the anaphylatoxin receptor C5L2 in response to LPS. Exogenous S1P restored C5L2 cell surface expression of Sphk1(-/-) mouse neutrophils to wild type levels but had no effect on cell surface expression of the other anaphylatoxin receptor, CD88. These results provide the first genetic evidence of the crucial role of Sphk1 in regulating the balance between expression of CD88 and C5L2 in phagocytes. S1P-mediated up-regulation of C5L2 is a novel therapeutic target for mitigating endotoxin-induced lung inflammatory injury.     

PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent and -independent effects

The G protein-coupled, receptor-activated phosphoinositide 3-kinase gamma (PI3Kgamma) mediates inflammatory responses and negatively controls cardiac contractility by reducing cAMP concentration. Here, we report that mice carrying a targeted mutation in the PI3Kgamma gene causing loss of kinase activity (PI3KgammaKD/KD) display reduced inflammatory reactions but no alterations in cardiac contractility. We show that, in PI3KgammaKD/KD hearts, cAMP levels are normal and that PI3Kgamma-deficient mice but not PI3KgammaKD/KD mice develop dramatic myocardial damage after chronic pressure overload induced by transverse aortic constriction (TAC). Finally, our data indicate that PI3Kgamma is an essential component of a complex controlling PDE3B phosphodiesterase-mediated cAMP destruction. Thus, cardiac PI3Kgamma participates in two distinct signaling pathways: a kinase-dependent activity that controls PKB/Akt as well as MAPK phosphorylation and contributes to TAC-induced cardiac remodeling, and a kinase-independent activity that relies on protein interactions to regulate PDE3B activity and negatively modulates cardiac contractility.     

Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.     

Role of C3a receptors, C5a receptors, and complement protein C6 deficiency in collagen antibody-induced arthritis in mice

The complement system, especially the alternative pathway, plays essential roles in the induction of injury in collagen Ab-induced arthritis (CAIA) in mice. The goal of the current study was to directly compare the roles of receptors for C3a and C5a, as well as the membrane attack complex, as effector mechanisms in the pathogenesis of CAIA. Clinical disease activity in C3aR(-/-), C5aR(-/-), and C6-deficient (C6-def) mice was decreased by 52, 94, and 65%, respectively, as compared with wild-type mice. Decreases in histopathologic injury as well as in IgG and C3 deposition paralleled the clinical disease activity. A decrease in the percentage of synovial neutrophils was observed in C3aR(-/-), C5aR(-/-), and C6-def mice, and a decrease in macrophages was observed in C3aR(-/-) and C5aR(-/-), but not in C6-def, mice. Synovial mRNA obtained by laser capture microdissection exhibited a decrease in TNF-α in C5aR(-/-) mice and in IL-1β in both C5aR(-/-) and C6-def mice, whereas C3aR(-/-) mice demonstrated no change in either cytokine. Our findings show that absent C3aR-, C5aR-, or membrane attack complex-initiated effector mechanisms each decrease susceptibility to CAIA, with clinical effects most pronounced in C5aR-deficient mice. Although the absence of C3aR, C5aR, or C6 led to differential deficiencies in effector mechanisms, decreased proximal joint IgG and C3 deposition was common to all three genotypes in comparison with wild-type mice. These data suggest the existence of positive-feedback amplification pathways downstream of all three effectors that promote additional IgG deposition and C3 activation in the joint.     

Phosphatidylinositol 3-kinase gamma signaling through protein kinase Czeta induces NADPH oxidase-mediated oxidant generation and NF-kappaB activation in endothelial cells

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kgamma in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kgamma-mediated oxidant signaling in the mechanism of NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110gamma catalytic subunit of PI3Kgamma. Tumor necrosis factor (TNF) alpha challenge of wild type ECs caused p110gamma translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110gamma-/- ECs. ROS production was the result of TNFalpha activation of Ser phosphorylation of NADPH oxidase subunit p47(phox) and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110gamma-/- ECs. Additionally, the TNFalpha-activated NF-kappaB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kgamma. TNFalpha challenge of p110gamma-/- ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCzeta. Thus, PI3Kgamma lies upstream of PKCzeta in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-kappaB activation and ICAM-1 expression.     

Defective dendritic cell migration and activation of adaptive immunity in PI3Kgamma-deficient mice

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.     

Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.     

Requirement for PI 3-kinase gamma in macrophage migration to MCP-1 and CSF-1

Phosphoinositide 3-kinases (PI3Ks) are important regulators of cell migration. The PI3K isoform gamma is primarily expressed in haematopoietic cells, and is activated by G protein-coupled receptors (GPCRs). Here, we investigate the contribution of PI3Kgamma to macrophage responses to chemoattractants, using bone marrow-derived macrophages from wild-type and PI3Kgamma-null mice. We observe that early membrane ruffling induced by MCP-1, which activates a GPCR, or by CSF-1, which activates a tyrosine kinase receptor, is unaltered in PI3Kgamma(-/-) mice, although by 30 min MCP-1-induced cell polarization was strongly reduced in PI3Kgamma(-/-) compared to wild-type macrophages. The migration behaviour of the macrophages was analysed by time-lapse microscopy in Dunn chemotaxis chambers. PI3Kgamma(-/-) macrophages showed reduced migration speed and translocation, and no chemotaxis to MCP-1. Interestingly, there was also a reduction in migration efficiency in PI3Kgamma(-/-) macrophages stimulated with CSF-1 although early CSF-1R signalling was normal. These results indicate that the initial actin reorganization induced by either a GPCR or tyrosine kinase receptor agonist is not dependent on PI3Kgamma, whereas PI3Kgamma is needed for optimal migration of macrophages to either agonist.     

Role of phosphatidylinositol 3-kinase-gamma in mediating lung neutrophil sequestration and vascular injury induced by E. coli sepsis

We addressed the in vivo role of phosphatidylinositol 3-kinase-gamma (PI3K-gamma) in signaling the sequestration of polymorphonuclear leukocytes (PMNs) in lungs and in the mechanism of inflammatory lung vascular injury. We studied mice with deletion of the p110 catalytic subunit of PI3K-gamma (PI3K-gamma(-/-) mice). We measured lung tissue PMN sequestration, microvascular permeability, and edema formation after bacteremia induced by intraperitoneal Escherichia coli challenge. PMN infiltration into the lung interstitium in PI3K-gamma(-/-) mice as assessed morphometrically was increased 100% over that in control mice within 1 h after bacterial challenge. PI3K-gamma(-/-) mice also developed a greater increase in lung microvascular permeability after E. coli challenge, resulting in edema formation. The augmented lung tissue PMN sequestration in PI3K-gamma(-/-) mice was associated with increased expression of the PMN adhesive proteins CD47 and beta(3)-integrins. We observed increased association of CD47 and beta(3)-integrins with the extracellular matrix protein vitronectin in lungs of PI3K-gamma(-/-) mice after E. coli challenge. PMNs from these mice also showed increased beta(3)-integrin expression and augmented beta(3)-integrin-dependent PMN adhesion to vitronectin. These results point to a key role of PMN PI3K-gamma in negatively regulating CD47 and beta(3)-integrin expression in gram-negative sepsis. PI3K-gamma activation in PMNs induced by E. coli may modulate the extent of lung tissue PMN sequestration secondary to CD47 and beta(3)-integrin expression. Therefore, the level of PI3K-gamma activation may be an important determinant of PMN-dependent lung vascular injury.     

Complement activation product C5a is a selective suppressor of TLR4-induced, but not TLR3-induced, production of IL-27(p28) from macrophages

There is accumulating evidence that the complement activation product, C5a, can orchestrate cellular immune functions. IL-27(p28/EBI3) is an emerging key player essential for regulating inflammatory responses and T cells. In this article, we report that C5a robustly suppressed IL-27(p28) gene expression and release in peritoneal macrophages. These cells from C57BL/6J mice abundantly produced IL-27(p28) after engagement of either the TLR3 (polyinosinic-polycytidylic acid) or TLR4 (LPS) receptor. Genetic deficiency of either TLR4 or LBP completely incapacitated the ability of macrophages to secrete IL-27(p28) in response to LPS. IL-27(p28)-producing macrophages also expressed the C5aR receptor, thus displaying an IL-27(p28)(+)F4/80(+)C5aR(+) phenotype. C5a suppressed IL-27(p28) in LPS-stimulated macrophages via interactions with the C5aR receptor rather than the C5L2 receptor. After endotoxemia, C5aR(-/-) mice displayed higher plasma levels of IL-27(p28) compared with C57BL/6J mice. C5a did not affect the release of IL-27(p28) or the frequency of IL-27(p28)(+)F4/80(+) macrophages after engagement of TLR3. Mechanistically, LPS activated both the NF-κB and the PI3K/Akt pathways, whereas C5a activated only the PI3K/Akt pathway. Engagement of PI3K/Akt was inhibitory for IL-27(p28) production, because PI3K/Akt pharmacologic blockade resulted in increased amounts of IL-27(p28) and reversed the suppressive effects of C5a. Blockade of PI3K/Akt in endotoxemic C57BL/6J mice resulted in higher generation of IL-27(p28). In contrast, the PI3K/Akt pathway was not involved in TLR3-mediated release of IL-27(p28). These data provide new evidence about how complement activation may selectively interfere with production of T cell regulatory cytokines by APCs in the varying contexts of either bacterial (TLR4 pathway) or viral (TLR3 pathway) infection.     

Evidence for anti-inflammatory effects of C5a on the innate IL-17A/IL-23 axis

There is growing evidence that the complement activation product C5a positively or negatively regulates inflammatory functions. The studies presented here report that C5a exerts anti-inflammatory effects by altering production of the cytokines IL-17A and IL-23 during endotoxic shock in young adult male C57BL/6J mice and has similar effects on macrophages from the same mice. IL-17A and IL-23 both appeared in plasma during endotoxemia, and their neutralization improved survival. The relevant sources of IL-17A during endotoxemia were not CD4(+) cells, γδ T cells, or NK cells but CD11b(+)F4/80(+) macrophages. The addition in vitro of C5a to lipopolysaccharide-activated peritoneal macrophages dose dependently antagonized the production of IL-17A (IC(50), 50-100 nM C5a) and IL-23 (IC(50), 10 nM C5a). This suppression required the receptor C5aR, but was independent of the second C5a receptor, C5L2. Genetic absence of C5aR was associated with much higher levels of IL-17A and IL-23 during endotoxic shock. Mechanistically, C5a mediated its effects on the IL-17A/IL-23 axis in a 2-step process. C5a caused activation of the PI3K-Akt and MEK1/2-ERK1/2 pathways, resulting in induction of IL-10, which powerfully inhibited production of IL-17A and IL-23. These data identify previously unknown mechanisms by which the anaphylatoxin C5a limits acute inflammation and antagonizes the IL-17A/IL-23 axis.     

Targeting phosphoinositide 3-kinase: moving towards therapy

Phosphoinositide 3-kinases (PI3K) orchestrate cell responses including mitogenic signaling, cell survival and growth, metabolic control, vesicular trafficking, degranulation, cytoskeletal rearrangement and migration. Deregulation of the PI3K pathway occurs by activating mutations in growth factor receptors or the PIK3CA locus coding for PI3Kalpha, by loss of function of the lipid phosphatase and tensin homolog deleted in chromosome ten (PTEN/MMAC/TEP1), by the up-regulation of protein kinase B (PKB/Akt), or the impairment of the tuberous sclerosis complex (TSC1/2). All these events are linked to growth and proliferation, and have thus prompted a significant interest in the pharmaceutical targeting of the PI3K pathway in cancer. Genetic targeting of PI3Kgamma (p110gamma) and PI3Kdelta (p110delta) in mice has underlined a central role of these PI3K isoforms in inflammation and allergy, as they modulate chemotaxis of leukocytes and degranulation in mast cells. Proof-of-concept molecules selective for PI3Kgamma have already successfully alleviated disease progress in murine models of rheumatoid arthritis and lupus erythematosus. As targeting PI3K moves forward to therapy of chronic, non-fatal disease, safety concerns for PI3K inhibitors increase. Many of the present inhibitor series interfere with target of rapamycin (TOR), DNA-dependent protein kinase (DNA-PK(cs)) and activity of the ataxia telangiectasia mutated gene product (ATM). Here we review the current disease-relevant knowledge for isoform-specific PI3K function in the above mentioned diseases, and review the progress of >400 recent patents covering pharmaceutical targeting of PI3K. Currently, several drugs targeting the PI3K pathway have entered clinical trials (phase I) for solid tumors and suppression of tissue damage after myocardial infarction (phases I,II).