It is well established that preexposure of human neutrophils to proinflammatory cytokines markedly augments the production of reactive oxygen species (ROS) to subsequent stimuli. This priming event is thought to be critical for localizing ROS to the vicinity of the inflammation, maximizing their role in the resolution of the inflammation, and minimizing the damage to surrounding tissue. We have used a new generation of isoform-selective phosphoinositide 3-kinase (PI3K) inhibitors to show that ROS production under these circumstances is regulated by temporal control of class I PI3K activity. Stimulation of tumor necrosis factor-␣ (TNF-␣)-primed human neutrophils with N-formylmethionyl-leucyl-phenylalanine (fMLP) results in biphasic activation of PI3K; the first phase is largely dependent on PI3K␥, and the second phase is largely dependent on PI3K␦. The second phase of PI3K activation requires the first phase; it is this second phase that is augmented by TNF-␣ priming and that regulates parallel activation of ROS production. Surprisingly, although TNF-␣-primed mouse bone marrow-derived neutrophils exhibit superficially similar patterns of PI3K activation and ROS production in response to fMLP, these responses are substantially lower and largely dependent on PI3K␥ alone. These results start to define which PI3K isoforms are responsible for modulating neutrophil responsiveness to infection and inflammation. IntroductionNeutrophils are critical components of the immune system and have a vital role in combating bacterial and fungal infections. 1 A key weapon in the neutrophil armory is the so-called "respiratory burst," the generation of reactive oxygen species (ROS) by a multicomponent oxidase complex. 2,3 Patients with chronic granulomatous disease (CGD) caused by defective expression of active oxidase components experience recurrent, life-threatening infections. 4 The role of ROS in fighting infections is complex. ROS are involved in the killing process directly through the damaging actions of oxygen radicals and their halogenated derivatives and indirectly through the activation of phagosomal proteases. [5][6][7] It is also becoming apparent that ROS may regulate the neutrophil lifespan, modify the extracellular matrix through which the neutrophils migrate, and modulate the function of other cells participating in the inflammatory response. [8][9][10][11][12] Given the potential for self-damage, a key feature of the inflammatory response is to confine ROS generation in time and space to areas where it is required. One way in which this is thought to occur is through a form of signal integration in which prior exposure to local proinflammatory factors is necessary for maximal activation by subsequent oxidase-triggering signals. [13][14][15][16][17] One of best studied examples of this "priming" phenomenon is the ability of tumor necrosis factor-␣ (TNF-␣), a cytokine released primarily by macrophages, to dramatically augment the oxidase response to bacterially derived peptides (eg, N-formyl-methionyl-leucylphenylal...

The interaction of interleukin 8 (IL-8) with heparin was studied by using synthetic IL-8 analogs with Cand N-terminal truncations. Elimination of the N-terminal region preceding the first cysteine, which constitutes the IL-8 receptor binding site, did not affect the afnity to heparinSepharose. Affinity, however, decreased with progressive truncation at the C terminus, and no binding was observed when the C-terminal a-helix was eliminated. The effect of heparin and other glycosaminoglycans on IL-8 activity was also tested.When IL-8 was applied together with heparan sulfate, neutrophil chemotaxis in vitro was enhanced up to 4-fold, and the stimulus-dependent increase in cytosolic free Ca2+ increased markedly in both rate and peak value. Heparin had a similar effect on the Ca2+ response but did not enhance chemotaxis. The glycosaminoglycans by themselves did not elicit neutrophil responses. Their enhancing effect was restricted to stimulation with IL-8 and was not observed when the unrelated chemoattractant fMet-Ile-Phe-Leu was used as the stimulus. Elastase released from stimulated neutrophils was inhibited by heparin, heparan sulfate, and, to a lesser extent, chondroitin sulfate B, conflrming previous observations. Taken together, these results suggest that heparan sulfate, which is present on the endothelial cell surface and in the basement membrane, may have a dual function in diapedesis, promotion of IL-8-dependent transmigration of neutrophils, and protection of the tissue microenvironment from damage by lytic enzymes released from the migrating cells.

MaterialA fter exposure to Ag via immunization or infection, B cells have the capacity to generate plasma cells and develop an extrafollicular response or, together with follicular dendritic cells, initiate the germinal center (GC) reaction (1). The GC is a microanatomical structure formed in B cell follicles in secondary lymphoid organs where Ag-specific B cells undergo division, isotype switching, somatic hypermutation, and differentiation into memory B cells or plasma cells. Cognate interaction between B and T cells in the GC is essential for the selection of high-affinity Ag-specific B cells, and access to T cell help is thought to be a limiting factor for the positive selection of GC B cells (2).The PI3K pathway has been implicated in lymphocyte development and activation. It transduces extracellular signals into the production of the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) by phosphorylation of phosphatidylinositol 4,5-bisphosphate. In lymphocytes, PIP 3 is generated by the class I PI3K catalytic subunits of which there are four isoforms named a, b, d, and g. The a, b, and d enzymes form heterodimers with one of five Src homology 2 domain-containing regulatory subunits termed p85a, p85b, p55g, p50a, and p55a, which mediate recruitment to phosphotyrosine-containing signalosomes (3). The levels of cellular PIP 3 are also regulated by phosphatases: phosphatase and tensin homolog deleted on chromosome 10 (PTEN) directly opposes PI3K by removing the 39 phosphate from PIP 3 ; the SHIP enzymes generate the second messenger, phosphatidylinositol 3,4-bisphosphate.Mice with germline mutations of the p110d catalytic subunit have demonstrated its importance for the GC reaction. However, it remains unclear whether this is a reflection of the requirement for p110d in B cells, T cells, dendritic cells (DCs), or other cell types required for the GC response (4, 5). B cells from p110d-deficient mice display in vitro defects in survival and proliferation that correlate with defective signal transduction following stimulation with Abs via the BCR or CD19 (6-8). Whether these in vitro defects are relevant in vivo is unclear since B cell-specific deletion of PTEN impaired class-switch recombination but not the magnitude of the GC response (9). T cell-specific deletion of the class IA regulatory subunits showed that the GC reaction and Ag-specific Ab titers were reduced, implicating a T cell-intrinsic requirement for class IA PI3K but leaving unresolved the nature of the relevant catalytic subunits (10). Using the p110d D910A mouse model, which carries a point mutation that renders p110d catalytically inactive, adoptive transfer experiments revealed impaired Th1/Th2 cytokine production and a 2-fold reduction in clonal expansion (5, 11). In contrast, T cell-specific deletion of PTEN removed the requirement for CD28 costimulation (12) and allowed enhanced IL-4 production (13). Gaining a better understanding of the cell intrinsic role for p110d in Ab responses should prove helpful in determining the mech...

Rac GTPases regulate cytoskeletal structure, gene expression, and reactive oxygen species (ROS) production. Rac2-deficient neutrophils cannot chemotax, produce ROS, or degranulate upon G protein-coupled receptor (GPCR) activation. Deficiency in PI3Kgamma, an upstream regulator of Rac, causes a similar phenotype. P-Rex1, a guanine-nucleotide exchange factor (GEF) for Rac, is believed to link GPCRs and PI3Kgamma to Rac-dependent neutrophil responses. We have investigated the functional importance of P-Rex1 by generating a P-Rex1(-/-) mouse. P-Rex1(-/-) mice are viable and healthy, with apparently normal leukocyte development, but with mild neutrophilia. In neutrophils from P-Rex1(-/-) mice, GPCR-dependent Rac2 activation is impaired, whereas Rac1 activation is less compromised. GPCR-dependent ROS formation is absent in lipopolysaccharide (LPS)-primed P-Rex1(-/-) neutrophils, but less affected in unprimed or TNFalpha-primed cells. Recruitment of P-Rex1(-/-) neutrophils to inflammatory sites is impaired. Surprisingly, chemotaxis of isolated neutrophils is only slightly reduced, with a mild defect in cell speed, but normal polarization and directionality. Secretion of azurophil granules is unaffected. In conclusion, P-Rex1 is an important regulator of neutrophil function by mediating a subset of Rac-dependent neutrophil responses. However, P-Rex1 is not an essential regulator of neutrophil chemotaxis and degranulation.

Collagen type II-induced arthritis (CIA) is an experimental model of arthritis that has been successfully used to dissect the pathogenesis of human rheumatoid arthritis and to identify potential therapeutic targets. We have used this model to evaluate the role of T cell co-stimulation in both disease development and progression. T cell co-stimulation is provided by ligation of CD28 with either B7-1 or B7-2 present on antigen-presenting cells and can be prevented by a soluble form of CTLA-4 (CTLA-4Ig) which binds with high affinity to both B7-1 and B7-2. We found that administration of CTLA-4Ig at the time of immunization prevented the development of CIA and was associated with lack of lymphocyte expansion within the draining lymph node and failure to produce anti-collagen IgG1 or IgG2a antibodies. To determine which CD28 ligand plays a more dominant role in CIA, we treated mice with monoclonal antibodies (mAb) against either B7-1 or B7-2. Neither anti-B7-1 nor anti-B7-2 had any effect on the course of CIA when given alone, but resulted in reduced incidence and clinical scores when given together. Interestingly, when treatment was delayed until after the onset of clinical disease, both CTLA-4Ig or anti-B7-1 plus anti-B7-2 mAb still ameliorated disease. Effective treatment was associated with a reduction in interferon-gamma production by lymph node cells following stimulation in vitro, suggesting that Th1 responses were diminished. This study points to a critical role of CD28 co-stimulation in the development and perpetuation of CIA in DBA/1 mice. Interestingly, it demonstrates an active role for T cells in the later stages of this disease and implicates both B7-1 and B7-2-mediated co-stimulation in the pathogenesis of CIA.

The role of PI3K activity in T lymphocyte development is obscure because mice deficient in single PI3K catalytic subunits either die before birth (p110α−/− and p110β−/−) or lack a significant T cell developmental phenotype (p110γ−/− and p110δ−/−). We have generated mice deficient in both p110γ and p110δ and show that p110γ/δ−/− mice have a profound block in T cell development that occurs at the β-selection checkpoint. We show that pre-TCR-induced signaling is significantly reduced in p110γ/δ−/− thymocytes and that this results in a concomitant lack of proliferative expansion and increased apoptosis. The survival defect in p110γ/δ−/− thymocytes is associated with increased levels of the pro-apoptotic molecule Bcl2 interacting mediator of cell death. This work demonstrates that PI3K activity is critical for T cell development and depends on the combined function of p110γ and p110δ.

The signal transduction pathways that lead activated natural killer (NK) cells to produce cytokines, releases cytotoxic granules, or do both, are not clearly dissected. For example, phosphoinositide 3-kinases (PI3Ks) are key players in the execution of both functions, but the relative contribution of each isoform is unknown. We show here that the catalytic isoform p110␦, not p110␥, was required for interferon-␥ (IFN-␥), tumor necrosis factor-␣ (TNF-␣), and granulocyte macrophage colony-stimulating factor (GM-CSF) secretion, whereas neither was necessary for cytotoxicity. Yet, when both p110␦ and p110␥ isoforms were inactivated by a combination of genetic and biochemical approaches, cytotoxicity was decreased. NK-cell numbers were also affected by the lack of p110␦ but not p110␥ and more severely so in mice lacking both subunits. These results provide genetic evidence that p110␦ is the dominant PI3K isoform for cytokine secretion by NK cells and suggest that PI3Ks cooperate during NK-cell development and cytotoxicity. ( IntroductionCellular cytotoxicity and production of inflammatory cytokines are the 2 main functions of natural killer (NK) cells. Although distinct cell subpopulations can specialize in one function rather than the other, 1-3 most NK cells develop the potential to do both. Multiple signal transduction pathways can activate NK cells, including some that are also involved in B and T lymphocyte stimulations. 4 Some of the pathways are independent of Syk-family kinases and revolve around phosphoinositide 3-kinases (PI3Ks). 5,6 PI3Ks are divided into 3 subclasses on the basis of their structure. Class I PI3Ks, which are the most studied in mammalian cell signaling, are further subdivided in 2 groups. Class IA consists of 5 isoforms of regulatory subunits (p85␣, p50␣, p55␣, p85␤, and p55␥) and 3 isoforms of catalytic subunits (p110␣, p110␤, and p110␦), which can interact in various combinations and are mostly activated downstream of tyrosine kinase-associated receptors for growth factors, antigens, cytokines, and costimulatory molecules. 7 Class IB includes only one regulatory isoform (p101) and one catalytic isoform (p110␥), and they are mostly activated downstream of G-coupled protein receptors. 8 P110␥ and p110␦ are expressed primarily in leukocytes, whereas p110␣ and p110␤ are expressed in all cells. The recruitment and activation of class I PI3Ks take place through adaptor molecules containing PI3K binding motif (YXXM), which interacts with SH2 domains of the regulatory subunits. Activated class I PI3Ks convert phosphatidylinositol-4,5-bisphospate (PIP 2 ) to phosphatidylinositol-3,4,5-trisphospate (PIP 3 ). 9 PIP 3 acts as a binding site for various intracellular molecules containing pleckstrin-homology (PH) domains that mediate crucial downstream effector functions. For example, AKT/PKB, 10 Vav, 11 BTK (Bruton agammaglobulinemia tyrosine kinase), ITK (IL-2-inducible T-cell kinase), 12 and PLC␥ (phospholipase C-␥) contain PH domains. Class II and III PI3Ks regulate vesicle trafficking, 13 but lit...

The guanosine triphosphatases (GTPases) of the immunity-associated protein (GIMAP) family of putative GTPases has been implicated in the regulation of T-lymphocyte development and survival. A mouse conditional knockout allele was generated for the immune GTPase gene