Protein Kinase Cζ (PKCζ) Regulates Ocular Inflammation and Apoptosis in Endotoxin-Induced Uveitis (EIU)
2007; Elsevier BV; Volume: 170; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2007.060236
ISSN1525-2191
AutoresYvonne de Kozak, B. Omri, Justine R. Smith, Marie‐Christine Naud, B. Thillaye–Goldenberg, Patricia Crisanti,
Tópico(s)NF-κB Signaling Pathways
ResumoWe show that inhibitory effect of interleukin-13 on endotoxin-induced uveitis in the Lewis rat is dependent on signaling activity of protein kinase Cζ (PKCζ). To understand the effect of interleukin-13 or PKCζ inhibitor treatment, the activation status of rat bone marrow-derived macrophages was studied in vitro. At 6 hours, lipopolysaccharide-stimulated macrophages produced tumor necrosis factor-α (TNF-α) with nuclear factor κB (NF-κB)/p65 expression. Treatment led to absence of NF-κB/p65 expression and low levels of TNF-α, suggesting accelerated inactivation of macrophages. At 24 hours after lipopolysaccharide stimulation, nuclear NF-κB/p65 decreased and nuclear NF-κB/p50 increased, associated with nuclear BCL-3 and a low level of TNF-α, indicating onset of spontaneous resolution. Treatment limited PKCζ cleavage, with expression of nuclear NF-κB/p50 and BCL-3 and low nuclear NF-κB/p65 promoting macrophage survival, as evidenced by Bcl-2 expression. At 24 hours, intraocular treatment decreased membranous expression of PKCζ by ocular cells, reduced vascular leakage with low nitric-oxide synthase-2 expression in vascular endothelial cells, and limited inflammatory cell infiltration with decreased intraocular TNF-α, interleukin-6, and nitric-oxide synthase-2 mRNA. Importantly, treatment decreased nuclear NF-κB/p65, increased transforming growth factor-β2, and reduced caspase 3 expression in infiltrating macrophages, implying a change of their phenotype within ocular microenvironment. Treatment accelerated endotoxin-induced uveitis resolution through premature apoptosis of neutrophils related to high expression of toll-like receptor 4 and caspase 3. We show that inhibitory effect of interleukin-13 on endotoxin-induced uveitis in the Lewis rat is dependent on signaling activity of protein kinase Cζ (PKCζ). To understand the effect of interleukin-13 or PKCζ inhibitor treatment, the activation status of rat bone marrow-derived macrophages was studied in vitro. At 6 hours, lipopolysaccharide-stimulated macrophages produced tumor necrosis factor-α (TNF-α) with nuclear factor κB (NF-κB)/p65 expression. Treatment led to absence of NF-κB/p65 expression and low levels of TNF-α, suggesting accelerated inactivation of macrophages. At 24 hours after lipopolysaccharide stimulation, nuclear NF-κB/p65 decreased and nuclear NF-κB/p50 increased, associated with nuclear BCL-3 and a low level of TNF-α, indicating onset of spontaneous resolution. Treatment limited PKCζ cleavage, with expression of nuclear NF-κB/p50 and BCL-3 and low nuclear NF-κB/p65 promoting macrophage survival, as evidenced by Bcl-2 expression. At 24 hours, intraocular treatment decreased membranous expression of PKCζ by ocular cells, reduced vascular leakage with low nitric-oxide synthase-2 expression in vascular endothelial cells, and limited inflammatory cell infiltration with decreased intraocular TNF-α, interleukin-6, and nitric-oxide synthase-2 mRNA. Importantly, treatment decreased nuclear NF-κB/p65, increased transforming growth factor-β2, and reduced caspase 3 expression in infiltrating macrophages, implying a change of their phenotype within ocular microenvironment. Treatment accelerated endotoxin-induced uveitis resolution through premature apoptosis of neutrophils related to high expression of toll-like receptor 4 and caspase 3. 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Because LPS-induced activation of cells occurs through TLR4, we investigated the expression of this receptor by infiltrating cells during EIU and in the presence of PKCζ inhibitor (PKCζi) or interleukin (IL)-13. We have previously shown that a single systemic or intraocular injection of IL-13 reduces the severity of EIU, with decreased expression of inflammatory cytokine and chemokine mRNA and reduced production of nitrite within the eye.39Marie O Thillaye-Goldenberg B Naud MC de Kozak Y Inhibition of endotoxin-induced uveitis and potentiation of local TNF-α and interleukin-6 mRNA expression by interleukin-13.Invest Ophthalmol Vis Sci. 1999; 40: 2275-2282PubMed Google Scholar, 40Lemaitre C Thillaye-Goldenberg B Naud MC de Kozak Y The effects of intraocular injection of interleukin-13 on endotoxin-induced uveitis in rats.Invest Ophthalmol Vis Sci. 2001; 42: 2022-2030PubMed Google Scholar IL-13, an anti-inflammatory cytokine produced by Th2 lymphocytes,41Minty A Chalon P Derocq JM Dumont X Guillemot JC Kaghad M Labit C Leplatois P Liauzun P Miloux B Minty C Casellas P Loison J Lupker J Shire D Ferrara P Caput D Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses.Nature. 1993; 362: 248-250Crossref PubMed Scopus (852) Google Scholar has been found to inhibit TNF-dependent in vitro activation of NF-κB and a second transcription factor, activation protein-1, and to block apoptosis of a number of human cell lines, activities that are presumably responsible for its immunosuppressive and anti-inflammatory effects.15Manna SK Aggarwal BB IL-13 suppresses TNF-induced activation of nuclear factor-κ B, activation protein-1, and apoptosis.J Immunol. 1998; 161: 2863-2872PubMed Google Scholar These effects are blocked by the PKC-specific inhibitor H-7, implicating PKC in IL-13 signaling. IL-13 has been shown to inhibit the PKC-triggered respiratory burst and to suppress NO release from macrophages.15Manna SK Aggarwal BB IL-13 suppresses TNF-induced activation of nuclear factor-κ B, activation protein-1, and apoptosis.J Immunol. 1998; 161: 2863-2872PubMed Google Scholar PKCζ appears to be essential for in vitro LPS-induced macrophage activation,42Cuschieri J Umanskiy K Solomkin J PKC-ζ is essential for endotoxin-induced macrophage activation.J Surg Res. 2004; 121: 76-83Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar and inhibition of the enzyme was associated with reduced TNF-α production. Macrophages play a critical role in ocular inflammation.43Pouvreau I Zech JC Thillaye-Goldenberg B Naud MC Van Rooijen N de Kozak Y Effect of macrophage depletion by liposomes containing dichloromethylene-diphosphonate on endotoxin-induced uveitis.J Neuroimmunol. 1998; 86: 171-181Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 44Hoey S Grabowski PS Ralston SH Forrester JV Liversidge J Nitric oxide accelerates the onset and increases the severity of experimental autoimmune uveoretinitis through an IFN-γ-dependent mechanism.J Immunol. 1997; 159: 5132-5142PubMed Google Scholar These nonspecific effector cells have a complex role, participating both in the process of tissue damage and in the resolution of inflammation.45Anderson CF Mosser DM A novel phenotype for an activated macrophage: the type 2 activated macrophage.J Leukoc Biol. 2002; 72: 101-106PubMed Google Scholar This prompted us to investigate the effect of IL-13 or PKCζi treatment on the expression of PKCζ and downstream signaling molecules responsible for macrophage activation in LPS-stimulated rat bone marrow-derived macrophages. We also analyzed the role of PKCζ in EIU and in the inhibition of this uveitis that is achieved by the administration of IL-13. We show that EIU treatment with IL-13 or PKCζi, induced the activation of signaling pathways involving PKCζ and NF-κB that regulate apoptosis or survival of resident ocular cells and infiltrating inflammatory cells. Male Lewis rats (Charles River, Saint-Aubin-les-Elbeuf, France), aged 8 weeks, were injected in one hind footpad with 500 μg/kg LPS from Salmonella typhimurium (Sigma Chemical Co., St. Louis, MO) in 0.1 ml of sterile pyrogen-free saline.39Marie O Thillaye-Goldenberg B Naud MC de Kozak Y Inhibition of endotoxin-induced uveitis and potentiation of local TNF-α and interleukin-6 mRNA expression by interleukin-13.Invest Ophthalmol Vis Sci. 1999; 40: 2275-2282PubMed Google Scholar This dose of LPS takes into account the weight of the animals and corresponds to 100 to 200 μg of LPS. Animals were housed in a 12-hour light and 12-hour dark cycle and fed water and dried ration ad libitum. A total of six separate experiments were performed, including a total of 59 rats. Rat tissues were separately processed for histopathological and immunohistochemical studies, reverse transcription-polymerase chain reaction analysis, and Western blot and other protein quantifications. Experimental protocols were developed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Anterior chamber injection was performed as previously described.40Lemaitre C Thillaye-Goldenberg B Naud MC de Kozak Y The effects of intraocular injection of interleukin-13 on endotoxin-induced uveitis in rats.Invest Ophthalmol Vis Sci. 2001; 42: 2022-2030PubMed Google Scholar In brief, rats were anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg Nembutal; Abbot, Saint-Remy sur Avre, France). Pupils were dilated by instillation of 1 drop of 5% tropicamide (Ciba Vision, Toulouse, France) and 1 drop of 1% tetracaine (Ciba Vision) was administered for local anesthesia. Four microliters of sterile pyrogen-free saline, PKCζi (PKCζ-specific inhibitory peptide, myr-SIYRRGARRWRKL, no. 539624, lot B42131; Calbiochem, San Diego, CA) and/or IL-13 (R&D, Abingdon, UK) was injected through the cornea into the anterior chamber under a surgical microscope, using a sterile syringe and 30-gauge needle (Microfine; Becton Dickinson, Meylan, France). The needle was left in the eye for 10 seconds to allow aqueous humor to leave the eye via the trabecular meshwork rather than by reflux along the needle track. The injection was performed near the apex of the cornea, taking care not to damage the iris or the lens. To investigate the role of PKCζ in transduction signals induced by LPS and to determine whether the inhibitory effect of IL-13 on EIU originated from an interaction with this signaling, different protocols were tested: intracameral injection of 4 μl of selected doses of PKCζi (3, 2, 1, 0.5 μg); intracameral injection of recombinant human IL-13 (2 ng); intracameral injection of PKCζi (1 μg) with IL-13 (1 ng); and saline as a negative control. All these injections were given simultaneously with the systemic injection of LPS. However, different time intervals of the PKCζi injection (3 hours before or 6 hours after LPS injection) were also tested. Intensity of the ocular inflammation was scored on a scale of 0 to 5. Animals were examined at a biomicroscope (slit lamp) by a masked investigator, and the degree of inflammation was scored at 24 hours after LPS injection, as previously described.12Yang P Smith JR Damodar KS Planck SR Rosenbaum JT Visualization of cell death in vivo during murine endotoxin-induced uveitis.Invest Ophthalmol Vis Sci. 2003; 44: 1993-1997Crossref PubMed Scopus (20) Google Scholar, 40Lemaitre C Thillaye-Goldenberg B Naud MC de Kozak Y The effects of intraocular injection of interleukin-13 on endotoxin-induced uveitis in rats.Invest Ophthalmol Vis Sci. 2001; 42: 2022-2030PubMed Google Scholar The level of protection was also expressed as percentage of protection, which was defined as: (EIU grade in saline-injected animals − EIU grade in PKCζi-injected animals)/(EIU grade in saline-injected animals). At the time of death, 24 hours after administration of PKCζi (3 μg in 4 μl) or saline and the LPS injection, aqueous humor and vitreous body were collected from both eyes of each animal and pooled. Protein concentration was determined in 1 μl of each sample using a Bradford assay with γ globulin as the standard (Bio-Rad Laboratories, Les Ulis, France). Enucleated eyes were fixed in 2% paraformaldehyde, embedded in OCT (Tissue-Tek; Miles Inc., Diagnostic Division, Elkhart, IN), and 10-μm anteroposterior cryostat sections were cut at the level of the optic nerve and stained using the following antibodies diluted 1:100, as described previously43Pouvreau I Zech JC Thillaye-Goldenberg B Naud MC Van Rooijen N de Kozak Y Effect of macrophage depletion by liposomes containing dichloromethylene-diphosphonate on endotoxin-induced uveitis.J Neuroimmunol. 1998; 86: 171-181Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar: mouse monoclonal anti-rat ED1 (macrophages and dendritic cells) (Serotec, Oxford, UK); rabbit polyclonal antibodies directed against PKCζ, NF-κB/p65, Bcl-2, and caspase 3 active form (described as detecting with a 50-fold preference for the active form compared with the inactive form), transforming growth factor (TGF)-β2 (SC-90) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit polyclonal antibody directed against inducible NOS (NOS-2; Transduction Laboratories, Lexington, KY); and goat polyclonal antibody anti-TLR4 antibody (SC-12511; Santa Cruz Biotechnology). Double immunostaining was performed as follows. Tissue sections were incubated overnight at 4°C with rabbit or goat polyclonal antibodies directed against the molecule of interest, followed the next day by a 1-hour incubation at room temperature with Alexa 488-conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR) or rabbit anti-goat IgG (H+L) antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), as appropriate, diluted 1:250. Subsequently, mouse monoclonal anti-rat ED1 was applied for 1 hour at room temperature followed by Alexa 594-conjugated goat anti-mouse IgG (H+L) (Molecular Probes), diluted 1:250. Control sections incubated without primary antibodies, or with addition of normal serum Ig in place of rabbit and goat polyclonal antibodies or mouse Ig of corresponding isotype in place of monoclonal mouse antibodies, were included in every staining run. Sections were mounted with an anti-fade medium with 4,6-diamidino-2-phenylindole (DAPI) or with propidium iodide (Vectashield; Vector laboratories, Burlingame, CA) and observed by fluorescence photomicroscopy (FXA, Microphot; Nikon, Melville, NY). Digitized micrographs were obtained using a digital camera (Spot; BFI Optilas, Evry, France). Apoptosis of cells was assessed with a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) kit (Roche, Indianapolis, IN) in strict accordance with the manufacturer's instructions. To quantify EIU and the effect of PKCζI treatment, all DAPI-positive inflammatory cells were counted across the entire ocular cross section, and cell number was expressed as the mean ± SEM of total cell number/animal as previously described.43Pouvreau I Zech JC Thillaye-Goldenberg B Naud MC Van Rooijen N de Kozak Y Effect of macrophage depletion by liposomes containing dichloromethylene-diphosphonate on endotoxin-induced uveitis.J Neuroimmunol. 1998; 86: 171-181Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar To determine the effect in EIU rats of the PKCζi treatment on the expression of specific signaling molecules by infiltrating inflammatory cells, dual immunostaining was performed as described above, using anti-PKCζ, anti-NF-κB, and anti-caspase 3 antibodies, as well as anti-ED1 antibody. ED1-negative inflammatory cells showed the characteristic morphological appearance of PMNs, with trilobed nuclei, after DAPI staining. Infiltrating inflammatory cells (ED1+ cells and PMNs) showing cytoplasmic or nuclear localization of PKCζ, NF-κB, and caspase 3 were counted in ocular tissues on cryostat sections from rats injected with LPS and treated with saline or with PKCζi (500 cells were counted per treatment). The percentage of nuclear or cytoplasmic staining was expressed as follows: 1) (number of ED1+ cells expressing specific marker in nucleus or cytoplasm)/(total number of ED1+ cells expressing the marker in the nucleus +
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