Surfactant Protein A Activation of Atypical Protein Kinase C ζ in IκB-α-Dependent Anti-Inflammatory Immune Regulation This information is current as Christina Moulakakis, Stefanie Adam, Ulrike Seitzer, Andra of September 29, 2021. B. Schromm, Michael Leitges and Cordula Stamme J Immunol 2007; 179:4480-4491; ; doi: 10.4049/jimmunol.179.7.4480 http://www.jimmunol.org/content/179/7/4480 Downloaded from References This article cites 61 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/179/7/4480.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Surfactant Protein A Activation of Atypical Protein Kinase C ␨ in I␬B-␣-Dependent Anti-Inflammatory Immune Regulation1 Christina Moulakakis,* Stefanie Adam,* Ulrike Seitzer,† Andra B. Schromm,‡ Michael Leitges,§ and Cordula Stamme2*¶ The pulmonary collectin surfactant protein (SP)-A has a pivotal role in anti-inflammatory modulation of lung immunity. The mechanisms underlying SP-A-mediated inhibition of LPS-induced NF-␬B activation in vivo and in vitro are only partially un- derstood. We previously demonstrated that SP-A stabilizes I␬B-␣, the primary regulator of NF-␬B, in alveolar macrophages (AM) both constitutively and in the presence of LPS. In this study, we show that in AM and PBMC from I␬B-␣ knockout/I␬B-␤ knockin mice, SP-A fails to inhibit LPS-induced TNF-␣ production and p65 nuclear translocation, confirming a critical role for I␬B-␣ in SP-A-mediated LPS inhibition. We identify atypical (a) protein kinase C (PKC) ␨ as a pivotal upstream regulator of SP-A- ؊ ؊ mediated I␬B-␣/NF-␬B pathway modulation deduced from blocking experiments and confirmed by using AM from PKC␨ / Downloaded from mice. SP-A transiently triggers aPKCThr410/403 phosphorylation, aPKC kinase activity, and translocation in primary rat AM. Coimmunoprecipitation experiments reveal that SP-A induces aPKC/p65 binding under constitutive conditions. Together the data indicate that anti-inflammatory macrophage activation via I␬B-␣ by SP-A critically depends on PKC␨ activity, and thus attribute a novel, stimulus-specific signaling function to PKC␨ in SP-A-modulated pulmonary immune response. The Journal of Immu- nology, 2007, 179: 4480–4491. http://www.jimmunol.org/ ulmonary surfactant is a lipid-protein complex that con- Alveolar macrophages (AM), normally accounting for ϳ95% of stitutes the alveolar liquid layer of the lung. The unique airspace leukocytes, are a unique class of professional phagocytes P composition of surfactant facilitates the functional combi- that represent the major effector cells of the pulmonary innate im- nation of different biological effects such as preventing alveolar mune system (6). SP-A directly interacts with AM through binding collapsing at expiration and immunomodulating innate pulmonary to cell surface receptors, resulting in modulation of chemotaxis, host defense responses (1, 2). The latter function is primarily me- phagocytosis, and modified pro- or anti-inflammatory immune re- diated by surfactant proteins (SP)3-A and SP-D belonging to the sponses (7). collectin family. SP-A, the most abundant pulmonary collectin, LPS, the main component of the outer leaflet of the outer by guest on September 29, 2021 binds and aggregates a variety of microorganisms and enhances membrane of Gram-negative bacteria, substantially contributes their phagocytosis and killing both in vivo and in vitro (3). SP-A- to the pathophysiology of the most frequent lung diseases, in- deficient mice exhibit delayed microbial clearance and higher lev- cluding pneumonia, acute lung injury, and acute respiratory dis- els of bronchoalveolar inflammatory mediators after intratracheal tress syndrome (8). Whereas LPS recognition benefits the host inoculation with a variety of clinically relevant pathogens (4), as by sensing bacteria and mobilizing defense mechanisms, an ex- well as to isolated LPS (5). aggerated response to LPS contributes to the development of a local or systemic septic shock syndrome. The interaction of picomolar concentrations of LPS with a receptor complex in- cluding TLR4, MD-2, LPS-binding protein, and CD14 on host *Department of Immunochemistry and Biochemical Microbiology, Division of Cel- cells initiates the sequential activation of multiple signaling lular Pneumology, Research Center Borstel, Leibniz Center for Medicine and Bio- ␬ science, Borstel, Germany; †Department of Immunology and Cell Biology, Division pathways, including NF- B, a central regulator of LPS-medi- of Veterinary Infection Biology and Immunology, Research Center Borstel, Leibniz ated cell activation (9–11). ‡ Center for Medicine and Bioscience, Borstel, Germany; Emmy-Noether Group Im- The most prominent NF-␬B heterodimer p65/p50 is sequestered munobiophysics, Research Center Borstel, Leibniz Center for Medicine and Bio- science, Borstel, Germany; §Biotechnolgy Centre of Oslo, University of Oslo, Oslo, in the cytoplasm by a family of inhibitory proteins, among Norway; and ¶Department of Anesthesiology, University Hospital of Lu¨beck, Lu¨- which I␬B-␣ is the best characterized and assumed to function beck, Germany as the primary regulator of NF-␬B in both stimulated and rest- Received for publication February 15, 2007. Accepted for publication July 30, 2007. ing cells (12, 13). In response to many stimuli including LPS, The costs of publication of this article were defrayed in part by the payment of page ␬ ␣ ␬ charges. This article must therefore be hereby marked advertisement in accordance I B- becomes degraded via ubiquitination in an I B kinase with 18 U.S.C. Section 1734 solely to indicate this fact. (IKK)-dependent manner (12, 14, 15). The removal of I␬B-␣ 1 This work was supported by the Deutsche Forschungsgemeinschaft 609/1-3 and allows the nuclear translocation of NF-␬B and subsequently the 609/1-4 (to C.S.) and 621/2-2 (to A.B.S.). transcription of downstream target genes, including I␬B-␣ itself 2 Address correspondence and reprint requests to Dr. Cordula Stamme, Research (16). Secondary clues point to an I␬B-independent pathway, Center Borstel, Parkallee 22, D-23845 Borstel, Germany. E-mail address: ␬ [email protected] and include the phosphorylation of p65 to activate NF- B- dependent gene transcription (17). 3 Abbreviations used in this paper: SP, surfactant protein; AKBI, I␬B-␣ knockout/ I␬B-␤ knockin; AM, alveolar macrophage; aPKC, atypical protein kinase C; cPKC, SP-A has been shown to inhibit LPS-induced TNF-␣ production classical protein kinase C; DAG, diacylglycerol; HI, heat inactivated; IKK, I␬B ki- (18–21), inducible NO synthase protein expression (22), NF-␬B ac- nase; PKC, protein kinase C; ps, pseudosubstrate; wt, wild type. tivity (20, 23, 24), and, upon diverse stimuli, NADPH oxidase (25) in Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 immunocompetent cells. We recently demonstrated that SP-A exerts www.jimmunol.org The Journal of Immunology 4481 its anti-inflammatory effects on LPS-challenged AM via a mechanism 37°C in a 5% CO2 atmosphere. Then the medium was changed and the involving a SP-A-mediated direct modulation of the basal and LPS- cells were treated with SP-A (20–60 ␮g/ml) and/or LPS (10–100 ng/ml) coupled I␬B-␣ turnover in these cells (24). In that study, SP-A in- for indicated times at 37°C in the presence of 0.2% heat-inactivated (HI) ␬ ␣ FCS. In separate experiments, AM were treated with a panel of kinase creased I B- protein expression in a dose- and time-dependent man- inhibitors to determine their possible effect on I␬B-␣ protein expression as ner without inducing I␬B-␣ phosphorylation or I␬B-␣ mRNA levels follows: Go¨-6976 (5 ␮M), Go¨-6850 (5 nM), chelerythrine chloride (6 and both basal and in the presence of LPS. However, the signaling path- 12 ␮M), or aPKCps peptides (2, 5, and 10 ␮M) were used to inhibit distinct ways controlling the SP-A-mediated attenuation of LPS-induced subsets of cPKC, novel PKC, or aPKCs. Wortmannin (50 nM) was used as PI3K inhibitor, and apigenin (30 ␮M) to inhibit protein kinase CKII. proinflammatory signals via I␬B-␣ remain unknown. A role for individual members of the protein kinase C (PKC) Nuclear protein extraction ␬ ␣ family in both SP-A immune modulation (26) and I B- turnover After treatment, cells were scraped off with 500 ␮l of cold PBS and spun (27) has been suggested previously. PKC family members are ex- at 4,500 ϫ g, 5 min, 4°C. The resulting pellet was resuspended in 400 ␮l pressed in many different cell types, in which they regulate a wide of ice-cold buffer A (10 mM Tris, 5 mM MgCl2,10mMKCl,1mM variety of cellular processes such as cell differentiation, cytoskel- ethyleneglycol-bis-(2-aminoethyl ether)-N,N,NЈ,NЈ-tetraacetic acid, 0.3 M ␤ etal remodeling, and gene expression in response to diverse stimuli sucrose, 1 mM DTT, 10 mM -glycerol phosphate, 0.5 mM PMSF, and 1.5 ␮l of protease inhibitor mixture (Complete; Roche)) and incubated on ice (28, 29). Based on sequence homology and the mechanism of their for 15 min; 25 ␮l of 10% Nonidet P-40 was then added and vortexed for regulation, individual members of the PKC family are subdivided 10 s.