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The Phosphatidylinositol 3-Kinase Pathway Selectively Controls sIL-1RA Not Interleukin-1β Production in the Septic Leukocytes*

  • Chris A. Learn
    Footnotes
    Affiliations
    Department of Medicine, Section of Infectious Diseases, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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  • M. Sean Boger
    Affiliations
    Department of Medicine, Section of Infectious Diseases, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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  • Liwu Li
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    Department of Medicine, Section of Infectious Diseases, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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  • Charles E. McCall
    Footnotes
    Affiliations
    Department of Medicine, Section of Infectious Diseases, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported by research funding from the NCI National Institutes of Health Training Grant CA-09422.
    ¶ Supported by National Institutes of Health Grant AI09169 and MM01 RR07122-10.
Open AccessPublished:January 01, 2001DOI:https://doi.org/10.1074/jbc.M100316200
      Microbial components such as bacterial endotoxin lipopolysaccharide (LPS) can trigger highly lethal septic shock. The cardinal features of septic leukocytes include the repressed production of inflammatory cytokines, such as interleukin-1 beta (IL-1β), and elevated production of anti-inflammatory cytokines, such as secretory interleukin-1 receptor antagonist (sIL-1RA). Pro- and anti-inflammatory cytokine gene transcriptions are equally repressed in septic leukocytes due to disruption of the LPS signaling pathway at the level of interleukin-1 receptor-associated kinase. The selective elevation of sIL-1RA protein in septic blood is caused by efficient translation of residual sIL-1RA message. In this study, we report that the LPS-inducible phosphatidylinositol 3-kinase (PI3-kinase)-dependent signaling pathway contributes to the elevated translation of sIL-1RA in septic/LPS-adapted leukocytes. We also observe that this pathway is gene specific and does not affect the production of proinflammatory IL-1β protein.
      LPS
      lipopolysaccharide
      IL
      interleukin
      sIL-1RA
      secretory interleukin-1 receptor antagonist
      TNF-α
      tumor necrosis factor-α
      TLR
      toll-like-receptor
      IRAK
      interleukin-1 receptor-associated kinase
      PI3
      phosphatidylinositol 3-kinase
      ELISA
      enzyme-linked immunosorbent assay
      PAGE
      polyacrylamide gel electrophoresis
      TBS
      Tris-buffered saline
      Sepsis occurs when inflammation induced by microbial infection spreads throughout the intravascular space of humans and animals causing failure of multiple organ-systems (reviewed in Ref.
      • Brun-Buiesson C.
      ). Sepsis has a mortality rate of 40–80 % and is the major cause of death in critical care units in this country. LPS1 plays a major role in inducing sepsis during infection caused by Gram-negative bacteria (
      • Wenzel R.P.
      • Pinsky M.R.
      • Ulevitch R.J.
      • Young L.
      ). Little improvement in the treatment of human sepsis has occurred since the syndrome was defined (
      • Bone R.C.
      ). The lack of knowledge of the altered innate immunity during sepsis may have contributed to the failures of treating sepsis (
      • Bone R.C.
      ).
      Blood leukocytes including monocytes, macrophages, and neutrophils are exquisitely responsive to microbial infection and play important roles in the induction of sepsis (
      • Stone R.
      ). The pathogenesis of sepsis during infection depends on induction of an autotoxic and apparently dysregulated inflammatory response from blood leukocytes including the activation of a number of genes with both pro- and anti-inflammatory effects (reviewed in 4). During the initial phase of bacterial infection, a transient induction of proinflammatory cytokines including IL-1β and tumor necrosis factor-α (TNF-α) occurs in blood leukocytes from septic patients. This initial phase is followed by a state of imbalance in which leukocytes have decreased production of proinflammatory cytokines and enhanced production of anti-inflammatory cytokines (
      • Bone R.
      • Grodzin C.
      • Balk R.
      ). In addition, ex vivo treatment with LPS does not stimulate IL-1β production in leukocytes from septic patients (
      • McCall C.E.
      • Grosso-Wilmoth L.M.
      • LaRue K.
      • Guzman R.N.
      • Cousart S.L.
      ). However, anti-inflammatory proteins, such as sIL-1RA (
      • Learn C.A.
      • Mizel S.B.
      • McCall C.E.
      ), are continually induced upon additional challenge with LPS in septic leukocytes. Such imbalance may lead to an adapted state of immunosuppression, thus increasing the mortality risk from subsequent super infection with other microorganisms. Recent reports in both clinical patient cases and animal models support this concept (
      • Bone R.
      • Grodzin C.
      • Balk R.
      ,
      • Cross A.
      • Asher L.
      • Seguin M.
      • Yuan L.
      • Kelly N.
      • Hammack C.
      • Sadoff J.
      • Gemski Jr., P.
      ). We refer to this adapted state as the septic leukocyte phenotype. The septic leukocyte phenotype can develop within hours following the onset of Gram-positive and Gram-negative infections and within 3 h when LPS is experimentally administered to humans or animals (
      • Van Der P.T.
      • Coyle S.M.
      • Moldawer L.L.
      • Lowry S.F.
      ). The septic leukocyte phenotype appears highly reproducible and displays consistent features that are sustained during the clinical course of septic shock in humans and animals (
      • McCall C.E.
      • Grosso-Wilmoth L.M.
      • LaRue K.
      • Guzman R.N.
      • Cousart S.L.
      ).
      The septic leukocyte phenotype has also been well documented in experimentally induced septic cell lines including human promonocytic THP-1 cells (
      • LaRue K.E.
      • McCall C.E.
      ) as well as the murine RAW264.7 and HeNC2 macrophage cells (
      • Medvedev A.E.
      • Kopydlowski K.M.
      • Vogel S.N.
      ). Upon initial LPS treatment, proinflammatory cytokines are rapidly induced within these cells. Mimicking human blood leukocytes from septic patients, prolonged treatment of THP-1 cells with LPS induces an adapted state as reflected by the suppression of proinflammatory proteins such as IL-1β and TNF-α. Upon further LPS treatment, LPS-adapted THP-1 cells exhibit continued production of anti-inflammatory proteins such as sIL-1RA (
      • Learn C.A.
      • Mizel S.B.
      • McCall C.E.
      ).
      Biochemical studies indicate that LPS-induced gene transcription accounts for the elevated cytokine production in both human blood leukocytes and model cell lines (
      • Yoza B.K.
      • Hu J.Y.
      • Cousart S.L.
      • McCall C.E.
      ). Cytokine gene transcription is repressed and no longer induced by LPS in septic blood leukocytes or LPS-adapted cell lines (
      • Yoza B.K.
      • Hu J.Y.
      • Cousart S.L.
      • McCall C.E.
      ).
      L. Mueller, L. Li, and C. E. McCall, submitted for publication.
      2L. Mueller, L. Li, and C. E. McCall, submitted for publication.
      Studies in our laboratory as well as in others indicate that the differential regulation of molecular signaling leads to an altered state of innate immunity in septic leukocytes. Toll-like-receptors (TLRs) have been shown in part to mediate LPS and other microbial-induced cytokine gene expression (reviewed in
      • Li L.
      • Cousart S.
      • Hu J.
      • McCall C.E.
      ). We observed that the interleukin-1 receptor-associated kinase (IRAK), which lies proximal in the LPS-TLR signaling pathway, is inactivated and reduced in quantity within 3 h of LPS stimulation. The alteration of IRAK is sustained for at least 17 h in the THP-1 cell line model of the septic leukocyte phenotype (
      • Li L.
      • Cousart S.
      • Hu J.
      • McCall C.E.
      ). Furthermore, a constitutive disruption of IRAK occurs in blood leukocytes of patients with sepsis.
      L. Li and C. E. McCall, unpublished observation.
      3L. Li and C. E. McCall, unpublished observation.
      It is likely that a disruption in IRAK signaling contributes to the repressed transcription of a set of LPS responsive genes, including proinflammatory IL-1β and TNF-α. Repression of transcription and rapid degradation of proinflammatory cytokine mRNAs contribute to decreased proinflammatory cytokine protein production in septic blood leukocytes and LPS-adapted cell lines.2 Despite the disrupted TLR signaling and reduced cytokine gene transcription, LPS can still induce sIL-1RA protein translation in adapted leukocytes.2 This indicates that the pathway controlling sIL-1RA translation is not disrupted and is responsive to further LPS challenge.
      In this investigation, we sought to identify the LPS signaling pathway(s) that are not interrupted and control translation of sIL-1RA in septic leukocytes. Using human blood from healthy donors and septic patients as well as the model THP-1 cell line, we observed that the PI3-kinase-dependent signaling pathway is still responsive to LPS in the septic leukocyte and selectively mediates LPS-induced translation of sIL-1RA but not IL-1β.

      DISCUSSION

      Two novel observations relevant to innate immunity and sepsis are provided by this study. First, despite repression of cytokine gene transcription due to disrupted TLR-IRAK-mediated signaling (
      • Li L.
      • Cousart S.
      • Hu J.
      • McCall C.E.
      ), a PI3-kinase-dependent pathway responsible for efficient sIL-1RA translation was selectively retained and remained responsive to further LPS challenge in the septic leukocytes. Second, the LPS-responsive PI3-kinase pathway selectively controlled sIL-1RA translation not IL-1β. Our study indicates that differential regulation of LPS-mediated signaling pathways contributes to altered cytokine protein profiles and the septic leukocyte phenotype (Fig.5).
      Figure thumbnail gr5
      Figure 5Schematic illustration of the altered signaling pathways in septic/adapted leukocytes.
      Our findings reveal a novel LPS signaling pathway that is responsive in LPS-adapted leukocytes. In normal leukocytes, LPS triggers TLR4 receptor-mediated signaling and activates a series of kinases including IRAK as well as various mitogen-activated protein kinases (
      • Medvedev A.E.
      • Kopydlowski K.M.
      • Vogel S.N.
      ,
      • Li L.
      • Cousart S.
      • Hu J.
      • McCall C.E.
      ). Prior exposure to LPS was reported to render leukocytes hyporesponsive to further LPS challenge, a phenomenon also known as endotoxin tolerance, which we refer to here as LPS adaptation. LPS-adapted leukocytes were shown not to respond to further LPS challenge as measured by suppression of IRAK kinase (
      • Li L.
      • Cousart S.
      • Hu J.
      • McCall C.E.
      ) and TLR4 signaling as well as all forms of mitogen-activated protein kinases (
      • Medvedev A.E.
      • Kopydlowski K.M.
      • Vogel S.N.
      ). Disruption of these LPS signaling events may account for the repressed cytokine gene transcription that occurs in septic leukocytes or experimentally LPS-adapted cell lines. However, LPS-adapted leukocytes still express several anti-inflammatory cytokines including IL-10 and sIL-1RA (
      • Learn C.A.
      • Mizel S.B.
      • McCall C.E.
      ). This indicates that endotoxin tolerance is not a total inhibition of cellular activities but rather an adaptation or reprogramming of cellular signaling events. Until now, it is not known which LPS signaling pathway still remains open and is responsible for the continued sIL-1RA production in LPS-adapted leukocytes. We show in this report that LPS induces PI3-kinase pathway activation in LPS-adapted cells (Fig. 3 C). The activation of PI3-kinase is commonly measured through the phosphorylation of endogenous Akt protein, a direct downstream target of PI3-kinase (reviewed in Ref.
      • Kandel E.S.
      • Hay N.
      ). PI3-kinase pathway activation and Akt phosphorylation was observed in LPS-stimulated normal macrophages, neutrophils, as well as monocytes (
      • Weinstein S.L.
      • Fin A.J.
      • Dave S.H.
      • Meng F.
      • Lowell C.A.
      • Sanghera J.S.
      • DeFranco A.L.
      ,
      • Diaz-Guerra M.J.
      • Castrillo A.
      • Martin-Sanz P.
      • Bosca L.
      ,
      • Bowling W.M.
      • Flye M.W.
      • Qiu Y.Y.
      • Callery M.P.
      ). However, there has been no previous biochemical study regarding the PI3-kinase activity in septic or LPS-adapted leukocytes. In this report, we are the first to report that Akt undergoes phosphorylation in LPS-adapted THP-1 cells upon further LPS treatment.
      In addition, our work provides a novel link between PI3-kinase pathway activation and sIL-1RA protein translation in septic/adapted leukocytes. The phospholipid products of PI3-kinase, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate, can act as second messengers and activate several downstream kinases including Akt, inositol phosphate kinase, and several calcium-dependent protein kinase C forms (
      • Leevers S.J.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      ). These downstream kinases can subsequently regulate multiple cellular events including gene transcription (
      • Reddy S.A.
      • Huang J.H.
      • Liao W.S.
      ) as well as protein translation (
      • Weinstein S.L.
      • Fin A.J.
      • Dave S.H.
      • Meng F.
      • Lowell C.A.
      • Sanghera J.S.
      • DeFranco A.L.
      ). Among the multiple targets of PI3-kinase, the activation of Akt, mTOR, and p70S6 kinases have been well documented to regulate ribosomal assembly and protein translational efficiency (
      • Sonenberg N.
      • Gingras A.C.
      ). We show here that inhibition of PI3-kinase by either wortmannin or LY294002 abrogates sIL-1RA protein production induced by LPS in septic whole blood or LPS-adapted THP-1 cells (Fig. 3, A and B). We observed that LPS-induced sIL-1RA message levels in human septic leukocytes or LPS-adapted THP-1 cells is not affected by PI3-kinase inhibitors (Fig. 4 A), suggesting that the PI3-kinase pathway selectively controls sIL-1RA translation not its transcription or message stability. We also observed that rapamycin, an inhibitor of mTOR, inhibits LPS-induced sIL-1RA protein production (data not shown). In addition, pulse-chase experiments show that inhibition of PI3-kinase significantly decreases incorporation of [35S]methionine into newly synthesized sIL-1RA protein (Fig. 4 B). Our findings indicate that the PI3-kinase pathway plays a critical role in directly regulating sIL-1RA translation.
      Interestingly, we observe that inhibition of PI3-kinase pathway does not interfere with other cytokine production such as IL-1β (Fig. 3,A and B). LPS-induced IL-1β message and protein levels in both septic blood and LPS-adapted THP-1 cells are not affected by PI3-kinase inhibitors. This indicates that the LPS-activated PI3-kinase pathway specifically regulates a certain set of cytokines and their translation. The mechanism of such regulation remains to be elucidated.
      Taken together, we conclude that septic/LPS-adapted leukocytes can still respond to LPS stimulation and undergo activation of PI3-kinase pathway. LPS-induced PI3-kinase pathway activation selectively contributes to the efficient translation of sIL-1RA not IL-1β. Our work further underscores that the septic leukocyte phenotype is a complex adaptation of cellular signaling events, involving not only repression of TLR-IRAK and several other signaling pathways but also selective activation of the PI3-kinase pathway. This altered phenotype may represent a modification of innate immunity that could either protect the host from further injury or produce an immunocompromised state. Further biochemical examination of the fine interplay of these signaling events is needed for better understanding and treatment of sepsis and other inflammatory diseases.

      Acknowledgments

      We thank Drs. Douglas Lyes and Steven Mizel for thoughtful discussions. Cell culture and media were supplied by the Tissue Culture Core Laboratory of Wake Forest University School of Medicine.

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