Hexokinase Is an Innate Immune Receptor for the Detection of Bacterial Peptidoglycan.

Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1ß and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor. PAPERCLIP.

Complement C5 inhibition protects against hemolytic anemia and acute kidney injury in anthrax peptidoglycan-induced sepsis in baboons.

Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of Bacillus anthracis, leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections.

Peptidoglycan induces disseminated intravascular coagulation in baboons through activation of both coagulation pathways.

Anthrax infections exhibit progressive coagulopathies that may contribute to the sepsis pathophysiology observed in fulminant disease. The hemostatic imbalance is recapitulated in primate models of late-stage disease but is uncommon in toxemic models, suggesting contribution of other bacterial pathogen-associated molecular patterns (PAMPs). Peptidoglycan (PGN) is a bacterial PAMP that engages cellular components at the cross talk between innate immunity and hemostasis. We hypothesized that PGN is critical for anthrax-induced coagulopathies and investigated the activation of blood coagulation in response to a sterile PGN infusion in primates. The PGN challenge, like the vegetative bacteria, induced a sepsis-like pathophysiology characterized by systemic inflammation, disseminated intravascular coagulation (DIC), organ dysfunction, and impaired survival. Importantly, the hemostatic impairment occurred early and in parallel with the inflammatory response, suggesting direct engagement of coagulation pathways. PGN infusion in baboons promoted early activation of contact factors evidenced by elevated protease-serpin complexes. Despite binding to contact factors, PGN did not directly activate either factor XII (FXII) or prekallikrein. PGN supported contact coagulation by enhancing enzymatic function of active FXII (FXIIa) and depressing its inhibition by antithrombin. In parallel, PGN induced de novo monocyte tissue factor expression in vitro and in vivo, promoting extrinsic clotting reactions at later stages. Activation of platelets further amplified the procoagulant state during PGN challenge, leading to DIC and subsequent ischemic damage of peripheral tissues. These data indicate that PGN may be a major cause for the pathophysiologic progression of Bacillus anthracis sepsis and is the primary PAMP behind the pathogen-induced coagulopathy in late-stage anthrax.

Inhibition of complement C5 protects against organ failure and reduces mortality in a baboon model of Escherichia coli sepsis.

Bacterial sepsis triggers robust activation of the complement system with subsequent generation of anaphylatoxins (C3a, C5a) and the terminal complement complex (TCC) that together contribute to organ failure and death. Here we tested the effect of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, using in vitro whole-blood assays and an in vivo baboon model of Escherichia coli sepsis. RA101295 strongly inhibited E. coli-induced complement activation both in vitro and in vivo by blocking the generation of C5a and the soluble form of TCC, sC5b-9. RA101295 reduced the E. coli-induced "oxidative burst," as well as leukocyte activation, without affecting host phagocytosis of E. coli RA101295 treatment reduced plasma LPS content in E. coli-challenged baboons, implying reduced complement-mediated bacteriolysis, whereas treated animals showed slightly improved bacterial clearance during the bacteremic stage compared with controls. Treatment with RA101295 also improved consumptive coagulopathy and preserved endothelial anticoagulant and vascular barrier functions. RA101295 abolished sepsis-induced surges in proinflammatory cytokines and attenuated systemic circulatory and febrile responses, likely reflecting decreased systemic levels of LPS and C5a. Overall, RA101295 treatment was associated with significant organ protection and markedly reduced mortality compared with nontreated controls (four of five animals survived in a 100% lethal model). We therefore conclude that inhibition of C5 cleavage during the bacteremic stage of sepsis could be an important therapeutic approach to prevent sepsis-induced inflammation, consumptive coagulopathy, and subsequent organ failure and death.

Serum Amyloid P and IgG Exhibit Differential Capabilities in the Activation of the Innate Immune System in Response to Bacillus anthracis Peptidoglycan.

We showed that human IgG supported the response by human innate immune cells to peptidoglycan (PGN) from Bacillus anthracis and PGN-induced complement activation. However, other serum constituents have been shown to interact with peptidoglycan, including the IgG-like soluble pattern recognition receptor serum amyloid P (SAP). Here, we compared the abilities of SAP and of IgG to support monocyte and complement responses to PGN. Utilizing in vitro methods, we demonstrate that SAP is superior to IgG in supporting monocyte production of cytokines in response to PGN. Like IgG, the response supported by SAP was enhanced by phagocytosis and signaling kinases, such as Syk, Src, and phosphatidylinositol 3-kinase, that are involved in various cellular processes, including Fc receptor signaling. Unlike IgG, SAP had no effect on the activation of complement in response to PGN. These data demonstrate an opsonophagocytic role for SAP in response to PGN that propagates a cellular response without propagating the formation of the terminal complement complex.

Gene expression profiling of primary human type I alveolar epithelial cells exposed to Bacillus anthracis spores reveals induction of neutrophil and monocyte chemokines.

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores must escape through the alveolar epithelial cell (AEC) barrier and migrate to regional lymph nodes, germinate and enter the circulatory system to cause disease. Several mechanisms to explain alveolar escape have been postulated, and all these tacitly involve the AEC barrier. In this study, we incorporate our primary human type I AEC model, microarray and gene enrichment analysis, qRT-PCR, multiplex ELISA, and neutrophil and monocyte chemotaxis assays to study the response of AEC to B. anthracis, (Sterne) spores at 4 and 24 h post-exposure. Spore exposure altered gene expression in AEC after 4 and 24 h and differentially expressed genes (±1.3 fold, p ≤ 0.05) included CCL4/MIP-1ß (4 h), CXCL8/IL-8 (4 and 24 h) and CXCL5/ENA-78 (24 h). Gene enrichment analysis revealed that pathways involving cytokine or chemokine activity, receptor binding, and innate immune responses to infection were prominent. Microarray results were confirmed by qRT-PCR and multiplex ELISA assays. Chemotaxis assays demonstrated that spores induced the release of biologically active neutrophil and monocyte chemokines, and that CXCL8/IL-8 was the major neutrophil chemokine. The small or sub-chemotactic doses of CXCL5/ENA-78, CXCL2/GROß and CCL20/MIP-3α may contribute to chemotaxis by priming effects. These data provide the first whole transcriptomic description of the human type I AEC initial response to B. anthracis spore exposure. Taken together, our findings contribute to an increased understanding of the role of AEC in the pathogenesis of inhalational anthrax.

Bacillus anthracis peptidoglycan activates human platelets through FcγRII and complement.

Platelet activation frequently accompanies sepsis and contributes to the sepsis-associated vascular leakage and coagulation dysfunction. Our previous work has implicated peptidoglycan (PGN) as an agent causing systemic inflammation in gram-positive sepsis. We used flow cytometry and fluorescent microscopy to define the effects of PGN on the activation of human platelets. PGN induced platelet aggregation, expression of the activated form of integrin αIIbß3, and exposure of phosphatidylserine (PS). These changes were dependent on immunoglobulin G and were attenuated by the Fcγ receptor IIa-blocking antibody IV.3, suggesting they are mediated by PGN-anti-PGN immune complexes signaling through Fcγ receptor IIa. PS exposure was not blocked by IV.3 but was sensitive to inhibitors of complement activation. PGN was a potent activator of the complement cascade in human plasma and caused deposition of C5b-9 on the platelet surface. Platelets with exposed PS had greatly accelerated prothrombinase activity. We conclude that PGN derived from gram-positive bacteria is a potent platelet agonist when complexed with anti-PGN antibody and could contribute to the coagulation dysfunction accompanying gram-positive infections.

Human SAP is a novel peptidoglycan recognition protein that induces complement-independent phagocytosis of Staphylococcus aureus.

The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer--a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an FcγR-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain-like receptors.

Anti-peptidoglycan antibodies and Fcγ receptors are the key mediators of inflammation in Gram-positive sepsis.

Gram-positive bacteria are an important public health problem, but it is unclear how they cause systemic inflammation in sepsis. Our previous work showed that peptidoglycan (PGN) induced proinflammatory cytokines in human cells by binding to an unknown extracellular receptor, followed by phagocytosis leading to the generation of NOD ligands. In this study, we used flow cytometry to identify host factors that supported PGN binding to immune cells. PGN binding required plasma, and plasma from all tested healthy donors contained IgG recognizing PGN. Plasma depleted of IgG or of anti-PGN Abs did not support PGN binding or PGN-triggered cytokine production. Adding back intact but not F(ab')2 IgG restored binding and cytokine production. Transfection of HEK293 cells with FcγRIIA enabled PGN binding and phagocytosis. These data establish a key role for anti-PGN IgG and FcγRs in supporting inflammation to a major structural element of Gram-positive bacteria and suggest that anti-PGN IgG contributes to human pathology in Gram-positive sepsis.

Toxin inhibition of antimicrobial factors induced by Bacillus anthracis peptidoglycan in human blood.

Here, we describe the capacity of Bacillus anthracis peptidoglycan (BaPGN) to trigger an antimicrobial response in human white blood cells (WBCs). Analysis of freshly isolated human blood cells found that monocytes and neutrophils, but not B and T cells, were highly responsive to BaPGN and produced a variety of cytokines and chemokines. This BaPGN-induced response was suppressed by anthrax lethal toxin (LT) and edema toxin (ET), with the most pronounced effect on human monocytes, and this corresponded with the higher levels of anthrax toxin receptor 1 (ANTXR1) in these cells than in neutrophils. The supernatant from BaPGN-treated cells altered the growth of B. anthracis Sterne, and this effect was blocked by LT, but not by ET. An FtsX mutant of B. anthracis known to be resistant to the antimicrobial effects of interferon-inducible Glu-Leu-Arg (ELR)-negative CXC chemokines was not affected by the BaPGN-induced antimicrobial effects. Collectively, these findings describe a system in which BaPGN triggers expression of antimicrobial factors in human WBCs and reveal a distinctive role, not shared with ET, in LT's capacity to suppress this response.

Cutting edge: primary innate immune cells respond efficiently to polymeric peptidoglycan, but not to peptidoglycan monomers.

The cell wall of bacteria induces proinflammatory cytokines in monocytes and neutrophils in human blood. The nature of the stimulating component of bacterial cell walls is not well understood. We have previously shown polymeric peptidoglycan (PGN) has this activity, and the cytokine response requires PGN internalization and trafficking to lysosomes. In this study, we demonstrate that peptidoglycan monomers such as muramyl dipeptide and soluble peptidoglycan fail to induce robust cytokine production in immune cells, although they activate the nucleotide-binding oligomerization domain proteins in transfected cell models. We further show that lysosomal extracts from immune cells degrade intact peptidoglycan into simpler products and that the lysosomal digestion products activate the nucleotide-binding oligomerization domain proteins. We conclude that naive innate immune cells recognize PGN in its polymeric form rather than monomers such as muramyl dipeptide and require PGN lysosomal hydrolysis to respond. These findings offer new opportunities in the treatment of sepsis, especially sepsis arising from Gram-positive organisms.

Gram-Positive Bacteria Cell Wall Peptidoglycan Polymers Activate Human Dendritic Cells to Produce IL-23 and IL-1ß and Promote TH17 Cell Differentiation.

Gram-positive bacterial infections are a major cause of organ failure and mortality in sepsis. Cell wall peptidoglycan (PGN) is shed during bacterial replication, and Bacillus anthracis PGN promotes a sepsis-like pathology in baboons. Herein, we determined the ability of polymeric Bacillus anthracis PGN free from TLR ligands to shape human dendritic cell (DC) responses that are important for the initiation of T cell immunity. Monocyte-derived DCs from healthy donors were incubated with PGN polymers isolated from Bacillus anthracis and Staphylococcus aureus. PGN activated the human DCs, as judged by the increased expression of surface HLA-DR, CD83, the T cell costimulatory molecules CD40 and CD86, and the chemokine receptor CCR7. PGN elicited the DC production of IL-23, IL-6, and IL-1ß but not IL-12p70. The PGN-stimulated DCs induced the differentiation of naïve allogeneic CD4+ T cells into T helper (TH) cells producing IL-17 and IL-21. Notably, the DCs from a subset of donors did not produce significant levels of IL-23 and IL-1ß upon PGN stimulation, suggesting that common polymorphisms in immune response genes regulate the PGN response. In sum, purified PGN is a highly stimulatory cell wall component that activates human DCs to secrete proinflammatory cytokines and promote the differentiation of TH17 cells that are important for neutrophil recruitment in extracellular bacterial infections.

IL-6 increases B-cell IgG production in a feed-forward proinflammatory mechanism to skew hematopoiesis and elevate myeloid production.

Src homology 2 domain-containing inositol 5-phosphatase (SHIP(-/-)) animals display an age-related increase in interleukin-6 (IL-6), a decrease in B lymphopoiesis, and an elevation in myelopoiesis. We investigated the origin of the IL-6 production and show that it is largely produced by peritoneal and splenic macrophages. IL-6 production by these macrophages is not a direct result of the loss of SHIP: IL-6 production is not spontaneous, is absent from bone marrow-derived macrophages, declines with prolonged culture of macrophages, and requires a stimulus present in vivo. The IL-6-rich peritoneal cavity of SHIP(-/-) mice shows more than 700-fold more immunoglobulin G (IgG) than wild-type, approximately 20% of which is aggregated or in an immune complex and contains B220(+) cells that secrete IgG. The SHIP-deficient peritoneal macrophages show evidence of IgG receptor stimulation. Animals lacking both the signal-transducing gamma-chain of IgG receptors and SHIP or Ig and SHIP produce less IL-6. The data indicate a feed-forward process in which peripheral macrophages, responding through IgG receptors to secreted IgG, produce IL-6, to support further B-cell production of IgG. Because of the proinflammatory phenotype of SHIP(-/-) animals, these findings emphasize the importance of IL-6-neutralizing strategies in autoimmune and proinflammatory diseases.

E-selectin engages PSGL-1 and CD44 through a common signaling pathway to induce integrin alphaLbeta2-mediated slow leukocyte rolling.

In inflamed venules, neutrophils rolling on E-selectin induce integrin alpha(L)beta(2)-dependent slow rolling on intercellular adhesion molecule-1 by activating Src family kinases (SFKs), DAP12 and Fc receptor-gamma (FcRgamma), spleen tyrosine kinase (Syk), and p38. E-selectin signaling cooperates with chemokine signaling to recruit neutrophils into tissues. Previous studies identified P-selectin glycoprotein ligand-1 (PSGL-1) as the essential E-selectin ligand and Fgr as the only SFK that initiate signaling to slow rolling. In contrast, we found that E-selectin engagement of PSGL-1 or CD44 triggered slow rolling through a common, lipid raft-dependent pathway that used the SFKs Hck and Lyn as well as Fgr. We identified the Tec kinase Bruton tyrosine kinase as a key signaling intermediate between Syk and p38. E-selectin engagement of PSGL-1 was dependent on its cytoplasmic domain to activate SFKs and slow rolling. Although recruiting phosphoinositide-3-kinase to the PSGL-1 cytoplasmic domain was reported to activate integrins, E-selectin-mediated slow rolling did not require phosphoinositide-3-kinase. Studies in mice confirmed the physiologic significance of these events for neutrophil slow rolling and recruitment during inflammation. Thus, E-selectin triggers common signals through distinct neutrophil glycoproteins to induce alpha(L)beta(2)-dependent slow rolling.

Interleukin-6 aborts lymphopoiesis and elevates production of myeloid cells in systemic lupus erythematosus-prone B6.Sle1.Yaa animals.

We previously reported the inhibitory action of interleukin-6 (IL-6) on B lymphopoiesis with SHIP(-/-) mice and showed that IL-6 biases lineage commitment toward myeloid cell fates in vitro and in vivo. Because elevated IL-6 is a feature of chronic inflammatory diseases, we applied an animal model of systemic lupus erythematosus (SLE) to determine whether IL-6 has similar effects on hematopoiesis. We found that IL-6 levels were elevated in the B6.Sle1.Yaa mice, and the increase was accompanied by losses of CD19(+) B cells and more primitive B-lymphoid progenitors in bone marrow. Both the CD19(+) B-cell population and their progenitors recovered in an IL-6(-/-) background. The uncommitted progenitors, containing precursors for both lymphoid and myeloid fates, expressed IL-6 receptor-alpha chain and responded to IL-6 by phosphorylation of STAT3. IL-6 stimulation caused uncommitted progenitors to express the Id1 transcription factor, which is known to inhibit lymphopoiesis and elevate myelopoiesis, and its expression was MAPK dependent. We conclude that chronic inflammatory conditions accompanied by increased IL-6 production bias uncommitted progenitors to a myeloid fate by inducing Id1 expression.

Resistance of human alveolar macrophages to Bacillus anthracis lethal toxin.

The etiologic agent of inhalational anthrax, Bacillus anthracis, produces virulence toxins that are important in the disease pathogenesis. Current studies suggest that mouse and human macrophages are susceptible to immunosuppressive effects of one of the virulence toxins, lethal toxin (LT). Thus a paradigm has emerged that holds that the alveolar macrophage (AM) does not play a significant role in the innate immune response to B. anthracis or defend against the pathogen as it is disabled by LT. This is inconsistent with animal models and autopsy studies that show minimal disease at the alveolar surface. We examined whether AM are immunosuppressed by LT. We found that human AM were relatively resistant to LT-mediated innate immune cytokine suppression, MEK cleavage, and induction of apoptosis as compared with mouse RAW 264.7 macrophages. Mouse AM and murine bone marrow-derived macrophages were also relatively resistant to LT-mediated apoptosis despite intermediate sensitivity to MEK cleavage. The binding component of LT, protective Ag, does not attach to human AM, although it did bind to mouse AM, murine bone marrow-derived macrophages, and RAW 264.7 macrophages. Human AM do not produce significant amounts of the protective Ag receptor anthrax toxin receptor 1 (TEM8/ANTXR1) and anthrax toxin receptor 2 (CMG2/ANTXR2). Thus, mature and differentiated AM are relatively resistant to the effects of LT as compared with mouse RAW 264.7 macrophages. AM resistance to LT may enhance clearance of the pathogen from the alveolar surface and explain why this surface is relatively free of B. anthracis in animal models and autopsy studies.

Vav and Rac activation in B cell antigen receptor endocytosis involves Vav recruitment to the adapter protein LAB.

The signal transduction events supporting B cell antigen receptor (BCR) endocytosis are not well understood. We have identified a pathway supporting BCR internalization that begins with tyrosine phosphorylation of the adapter protein LAB. Phosphorylated LAB recruits a complex of Grb2-dynamin and the guanine nucleotide exchange factor Vav. Vav is required for activation of the small GTPases Rac1 and Rac2. All these proteins contribute to (and dynamin, Vav, and Rac1/2 are required for) BCR endocytosis and presentation of antigen to T cells. This is the first description of a sequential signal transduction pathway from BCR to internalization and antigen presentation.

Inflammatory cytokine response to Bacillus anthracis peptidoglycan requires phagocytosis and lysosomal trafficking.

During advanced stages of inhalation anthrax, Bacillus anthracis accumulates at high levels in the bloodstream of the infected host. This bacteremia leads to sepsis during late-stage anthrax; however, the mechanisms through which B. anthracis-derived factors contribute to the pathology of infected hosts are poorly defined. Peptidoglycan, a major component of the cell wall of Gram-positive bacteria, can provoke symptoms of sepsis in animal models. We have previously shown that peptidoglycan of B. anthracis can induce the production of proinflammatory cytokines by cells in human blood. Here, we show that biologically active peptidoglycan is shed from an active culture of encapsulated B. anthracis strain Ames in blood. Peptidoglycan is able to bind to surfaces of responding cells, and internalization of peptidoglycan is required for the production of inflammatory cytokines. We also show that the peptidoglycan traffics to lysosomes, and lysosomal function is required for cytokine production. We conclude that peptidoglycan of B. anthracis is initially bound by an unknown extracellular receptor, is phagocytosed, and traffics to lysosomes, where it is degraded to a product recognized by an intracellular receptor. Binding of the peptidoglycan product to the intracellular receptor causes a proinflammatory response. These findings provide new insight into the mechanism by which B. anthracis triggers sepsis during a critical stage of anthrax disease.

Src homology 2-containing 5-inositol phosphatase (SHIP) suppresses an early stage of lymphoid cell development through elevated interleukin-6 production by myeloid cells in bone marrow.

The Src homology (SH)2-containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP(-/-) animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP(-/-) mice. Lineage-negative marrow (Lin(-)) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP(-/-) mice. Furthermore, culture supernatants of SHIP(-/-) Lin(-) cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP(-/-) Lin(-) cells contained more IL-6 transcripts than wild-type Lin(-) cells, and neutralizing anti-IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP(-/-) Lin(-) cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin(-) bone marrow cells reproduced the phenotype of SHIP(-/-) bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow.

Human lung innate immune cytokine response to adenovirus type 7.

Adenovirus (Ad) type 7 can cause severe infection, including pneumonia, in military recruits and children. The initial inflammation is a neutrophilic interstitial infiltration with neutrophilic alveolitis. Subsequently, monocytes become evident and, finally, there is a predominantly lymphocytic infiltrate. We have established that Ad7 infection of epithelial cells stimulates release of the neutrophil chemotaxin interleukin (IL)-8, and have extended these studies to a human lung tissue model. Here, we studied cytokine responses to Ad7 in human alveolar macrophages (HAM) and our human lung tissue model. Both ELISA and RNase-protection assay (RPA) data demonstrated that, upon Ad7 infection, IP-10 and MIP-1alpha/beta are released from HAM. IP-10 and MIP-1alpha/beta protein levels were induced 2- and 3-fold, respectively, in HAM 24 h after Ad7 infection. We then investigated induction of specific cytokines in human lung tissue by RPA and ELISA. The results showed that IL-8 and IL-6 were induced 8 h after infection and, by 24 h, levels of IL-8, IL-6, MIP-1alpha/beta and MCP-1 were all increased. IP-10, a monocyte and lymphocyte chemokine, was also induced 30-fold, but only 24 h after infection. Immunohistochemistry staining confirmed that IL-8 was only released from the epithelial cells of lung slices and not from macrophages. IP-10 was secreted from both macrophages and epithelial cells. Moreover, full induction of IP-10 is likely to require participation and cooperation of both epithelial cells and macrophages in intact lung. Understanding the cytokine and chemokine induction during Ad7 infection may lead to novel ways to modulate the response to this pathogen.