Skip to main content

Main menu

  • Home
  • Articles
    • Current Issue
    • Next in The JI
    • Archive
    • Brief Reviews
      • Neuroimmunology: To Sense and Protect
    • Pillars of Immunology
    • Translating Immunology
    • Most Read
    • Top Downloads
    • Annual Meeting Abstracts
  • COVID-19/SARS/MERS Articles
  • Info
    • About the Journal
    • For Authors
    • Journal Policies
    • Influence Statement
    • For Advertisers
  • Editors
  • Submit
    • Submit a Manuscript
    • Instructions for Authors
    • Journal Policies
  • Subscribe
    • Journal Subscriptions
    • Email Alerts
    • RSS Feeds
    • ImmunoCasts
  • More
    • Most Read
    • Most Cited
    • ImmunoCasts
    • AAI Disclaimer
    • Feedback
    • Help
    • Accessibility Statement
  • Other Publications
    • American Association of Immunologists
    • ImmunoHorizons

User menu

  • Subscribe
  • Log in

Search

  • Advanced search
The Journal of Immunology
  • Other Publications
    • American Association of Immunologists
    • ImmunoHorizons
  • Subscribe
  • Log in
The Journal of Immunology

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Next in The JI
    • Archive
    • Brief Reviews
    • Pillars of Immunology
    • Translating Immunology
    • Most Read
    • Top Downloads
    • Annual Meeting Abstracts
  • COVID-19/SARS/MERS Articles
  • Info
    • About the Journal
    • For Authors
    • Journal Policies
    • Influence Statement
    • For Advertisers
  • Editors
  • Submit
    • Submit a Manuscript
    • Instructions for Authors
    • Journal Policies
  • Subscribe
    • Journal Subscriptions
    • Email Alerts
    • RSS Feeds
    • ImmunoCasts
  • More
    • Most Read
    • Most Cited
    • ImmunoCasts
    • AAI Disclaimer
    • Feedback
    • Help
    • Accessibility Statement
  • Follow The Journal of Immunology on Twitter
  • Follow The Journal of Immunology on RSS

Cyclooxygenase-2 Expression in Macrophages: Modulation by Protein Kinase C-α

Mélanie Giroux and Albert Descoteaux
J Immunol October 1, 2000, 165 (7) 3985-3991; DOI: https://doi.org/10.4049/jimmunol.165.7.3985
Mélanie Giroux
Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Albert Descoteaux
Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high levels of PG production during inflammation and immune responses. Previous studies with pharmacological inhibitors suggested a role for protein kinase C (PKC) in PG production possibly by regulating COX-2 expression. In this study, we addressed the role of PKC-α in the modulation of COX-2 expression and PGE2 synthesis by the overexpressing of a dominant-negative (DN) mutant of this isoenzyme in the mouse macrophage cell line RAW 264.7. We investigated the effect of various stimuli on COX-2 expression, namely, LPS, IFN-γ, and the intracellular parasite Leishmania donovani. Whereas LPS-induced COX-2 mRNA and protein expression were down-regulated in DN PKC-α-overexpressing clones, IFN-γ-induced COX-2 expression was up-regulated in DN PKC-α-overexpressing clones with respect to normal RAW 264.7 cells. Measurements of PGE2 levels revealed a strong correlation between PGE2 secretion and IFN-γ-induced COX-2 mRNA and protein levels in DN PKC-α-overexpressing clones. Taken together, these results suggest a role for PKC-α in the modulation of LPS- and IFN-γ-induced COX-2 expression, as well as in IFN-γ-induced PGE2 secretion.

Prostaglandins are important mediators of inflammatory and immune responses. Their secretion is induced by various stimuli including LPS, phorbol esters, cytokines, and phagocytosis (1). PGE2 is one of the main PGs secreted in large quantities by macrophages and acts as an autocrine regulator of their activity (2, 3). Cyclooxygenases (COX),3 the key enzymes responsible for the conversion of arachidonic acid to PGs, exist in two isoforms with different physiological functions. Whereas COX-1 is constitutively expressed in most cell types and is responsible for regulating normal physiological functions (3, 4), COX-2 is inducible in cells playing a role in inflammation such as macrophages, fibroblasts, and endothelial cells (5, 6). In human and murine macrophages, COX-2 expression is induced by LPS, IL-1, and phorbol esters (6, 7, 8, 9). Studies with the murine macrophage cell line RAW 264.7 indicated that accumulation of COX-2 mRNA can be induced by a combination of IFN-γ and LPS but not by IFN-γ alone (10). In addition to soluble mediators, pathogens such as the intracellular parasite Leishmania donovani can increase synthesis of PGE2, possibly by inducing alterations in the COX pathway (11, 12).

Previous studies using protein kinase C (PKC) inhibitors and activators suggested that PGE2 synthesis requires the activation of PKC in the mouse macrophage cell line RAW 264.7, as well as in peritoneal macrophages (1, 13). Twelve isoenzymes of PKC, a family of protein serine/threonine kinases, have been identified so far. Differences in their structure, requirement for activity, subcellular localization, and substrate specificity suggest that in a given cell, the various PKC isoenzymes may exert specific functions. Six of them are expressed in macrophages but their respective roles in the regulation of macrophage functions are poorly understood (14, 15). Using clones of the RAW 264.7 macrophage cell line overexpressing a dominant-negative (DN) mutant of PKC-α (DN PKC-α), we recently reported that PKC-α regulates selective LPS-induced responses, including inducible NO synthase (iNOS) and IL-1α expression (16). This study led us to propose a role for PKC-α in the regulation of inflammatory responses. Previous studies based on selective depletion of PKC isoenzymes and their differential sensitivities to pharmacological inhibitors led to the suggestion that PKC-α regulates zymosan-stimulated arachidonic acid metabolism and eicosanoid synthesis in peritoneal macrophages (17). To further investigate the role of PKC-α in the regulation of COX-2 expression and PGE2 secretion, we used DN PKC-α-overexpressing clones of the RAW 264.7 macrophage line (16). We obtained evidence that PKC-α modulates COX-2 expression in macrophages exposed to both LPS and IFN-γ, thereby providing additional evidence that PKC-α is involved in the regulation of macrophage inflammatory responses.

Materials and Methods

Cell lines

The murine macrophage cell line RAW 264.7 transfected with the expression vector pCIN-4, and the DN PKC-α-overexpressing clones B1 and C2 (16) were cultured in a 37°C incubator with 5% CO2 in DMEM with glutamine (Life Technologies, Ontario, Canada), containing 10% heat-inactivated FBS (HyClone, Logan, UT), 10 mM HEPES pH 7.3, and antibiotics supplemented with 200 μg/ml G418 (Life Technologies).

Bone marrow-derived macrophages (BMM)

BMM were obtained as previously described (18). Briefly, bone marrow cells obtained from femurs of 6- to 8-wk-old female BALB/c mice (Charles River, St-Constant, Québec, Canada), were freed of RBC by osmotic shock and resuspended in complete medium with 15% (v/v) L929 cell-conditioned medium. After 1 day in culture (37°C, 5% CO2), nonadherent cells were transferred into new culture dishes and then allowed to differentiate and adhere for 6 days. BMM were made quiescent by culturing them in CSF-1-free medium for 18 h before being used.

L. donovani

Promastigotes of L. donovani (Ethiopian strain LV9, obtained from G. Matlashewski, McGill University, Montréal, QC, Canada) were freshly derived from amastigotes isolated from the spleen of an infected hamster and were grown in at 26°C in RPMI 1640 supplemented with 20% heat-inactivated FBS, 100 μM adenine, 5 μM hemin, 1 μM biotin, 20 mM 2-[N-morpholino]ethanesulfonic acid, pH 5.5, and antibiotics. For infections with L. donovani, 2.5 × 106 adherent macrophages were incubated with 2.5 × 107 parasites for 8 h.

Northern blot analyses

Total RNA preparation and Northern blot analyses were performed essentially as described previously (16, 19). The probe for murine COX-2 consisted of the 1.2-kb EcoRI/ApaI fragment from COX-2 cDNA amplified by PCR using oligodeoxynucleotides AD-24 (5′-CCCCTTCCTGCGAAGTTTAATC-3′) and AD-25 (5′-GCATCTGGACGAGGTTTTTCC-3′).

Plasmids

The luciferase reporter vector (pTIS10L) containing the promoter region of the mouse COX-2 gene (20) (−963/+70 from the transcription initiation site) was provided by Harvey Herschman (University of California, Los Angeles, CA) and was used for transient transfections studies. The PKC-α expression vector (pCMV-PKC-α) was constructed by insertion of the human wild-type PKC-α cDNA (21) into the HindIII site of the expression vector pRcCMV (Invitrogen, San Diego, CA) and was used for overexpression analyses. The pRL-TK plasmid encoding the Renilla luciferase was obtained from Promega (Madison, WI).

Transient transfections

Adherent cells (2.5 × 105/well) were transfected using GenePorter (Gene Therapy Systems, San Diego, CA) with 0.25 μg of COX-2 luciferase reporter plasmid, and either 0.65 μg of pRcCMV (Invitrogen) or pCMV-PKC-α expression vector. All transfections included 0.1 μg of pRL-TK (Promega) as transfection efficiency control. Cells were transfected with 250 μl DNA/GenePorter mix for 5 h, and 1 ml of serum-free medium was added. Cells were treated 7 h later with 100 ng/ml LPS and harvested at 12 h in Reporter lysis buffer (Promega). Firefly and Renilla luciferase values were obtained by analyzing 20 μl of cell extracts according to standard instructions provided in the Dual Luciferase kit (Promega) using a Lumat LB 9507 luminometer (EG & G Berthold, Nashua, NH). Statistically significant differences were identified using the unpaired Student’s t test. Values of p = 0.01 were considered statistically significant.

Western blot analyses

Western blot analyses were performed as described previously (16). Anti-COX-2 mAbs were obtained from Transduction Laboratories (Lexington, KY).

PGE2 production

PGE2 levels in the supernatants of macrophage were measured by competitive immunoassay (EIA; Cayman Chemicals, Ann Arbor, MI) after 8 h of incubation with different stimuli as recommended by the manufacturer. When indicated, the COX inhibitors NS398 (5 μM) and valeryl salicylate (1 mM) (Cayman Chemicals) or the iNOS inhibitor NG-monomethyl-l-arginine monoacetate (l-NMMA; 500 μm) (Alexis, San Diego, CA) were used. Statistically significant differences were identified using the unpaired Student’s t test. Values of p = 0.01 were considered statistically significant.

Results

Effect of DN PKC-α overexpression on LPS-induced COX-2 expression

In macrophages, COX-2 expression is strongly induced by LPS, phorbol-ester, and several cytokines (22). To investigate the role of PKC-α in this process, we measured COX-2 mRNA accumulation and protein level expression in normal RAW 264.7 cells (containing the empty vector) and in DN PKC-α-overexpressing clones (B1 and C2; Ref. 16) after stimulation with LPS (10 and 100 ng/ml) for 8 h. In normal RAW 264.7 cells, LPS induced the expression of COX-2 mRNA accumulation and protein synthesis in a dose-dependent manner (Fig. 1⇓, A and B, lanes 1–3). In DN PKC-α-overexpressing clones, LPS-induced COX-2 mRNA accumulation, and protein levels were significantly inhibited. Densitometric analyses revealed that in clone B1, COX-2 mRNA levels were reduced by 10- to 20-fold (Fig. 1⇓A, lanes 4–6), and protein levels were reduced by ∼4-fold (Fig. 1⇓B, lanes 4–6) with respect to the levels observed in control cells. In clone C2, LPS-induced COX-2 mRNA levels were barely detectable (Fig. 1⇓A, lanes 7–9), whereas COX-2 protein levels were reduced by ∼3- to 5-fold with respect to control cells (Fig. 1⇓B, lanes 7–9). Thus, similar to LPS-induced IL-1α and iNOS expression (16), DN PKC-α overexpression strongly inhibited LPS-induced COX-2 expression in RAW 264.7 macrophages.

FIGURE 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 1.

Effect of DN PKC-α overexpression on LPS-induced COX-2 expression. Adherent cells (vector alone, clone B1, and clone C2) were incubated in the absence (lanes 1, 4, and 7) or in the presence of either 10 ng/ml (lanes 2, 5, and 8) or 100 ng/ml (lanes 3, 6, and 9) LPS for 8 h. Total RNA was extracted and Northern blot analyses was performed (A), and cell extracts were prepared for Western blot analyses (B) as described in Materials and Methods. RNA integrity and loading were assessed by ethidium bromide staining. Similar results were obtained in at least three separate experiments.

Overexpression of PKC-α increases LPS-induced COX-2 promoter activity

The inhibition of LPS-induced COX-2 expression in DN PKC-α-overexpressing macrophages indicated that PKC-α plays a role in modulating COX-2 expression. To further demonstrate the involvement of PKC-α in the induction of COX-2 by LPS, we transiently transfected RAW 264.7 cells with a COX-2-luciferase reporter and a wild-type PKC-α expression vector. Overexpression of wild-type PKC-α had no effect on basal COX-2 promoter activity in untreated RAW 264.7 cells (Fig. 2⇓). In contrast, PKC-α overexpression significantly increased LPS-stimulated COX-2 promoter activity by ∼2-fold with respect to controls (Fig. 2⇓, p = 0.0001, n = 3). These data are consistent with PKC-α playing a role in modulating COX-2 expression in LPS-stimulated macrophages.

FIGURE 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 2.

Overexpression of PKC-α increases LPS-induced COX-2 promoter activity in RAW 264.7 macrophages. Adherent RAW 264.7 cells were transiently transfected with the COX-2/Luc reporter construct and pRL-TK for 5 h along with either the control vector (▨) or the wild-type PKC-α expression vector (□). Cells were incubated for 7 h and then stimulated with 100 ng/ml LPS for 12 h. Firefly and Renilla luciferase activities were determined in cell extracts. Data are expressed as a ratio of firefly luciferase value/Renilla luciferase value. Experiments were performed in triplicate and are representative of results obtained in two separate experiments. ∗∗, p = 0.0001 as compared with LPS-stimulated cells transfected with control vector.

Effect of DN PKC-α overexpression on COX-2 expression following a stimulation with IFN-γ

IFN-γ is a potent regulator of macrophage function (23). In addition to inducing the expression of several genes, incubation of macrophages with IFN-γ enhances their responsiveness to LPS (10, 24). To determine whether PKC-α plays a role in the regulation of IFN-γ-induced responses, we have measured the induction of COX-2 mRNA accumulation and protein synthesis in control RAW 264.7 cells and in the DN PKC-α-overexpressing clones B1 and C2 in response to 100 U/ml IFN-γ alone or in combination with 100 ng/ml LPS. Macrophages were primed with 100 U/ml IFN-γ for 18 h before the addition of either 100 U/ml IFN-γ or the combination of 100 U/ml IFN-γ and 100 ng/ml LPS. IFN-γ induced an important increase of COX-2 mRNA accumulation in DN PKC-α-overexpressing cells (20-fold for clone B1 and 60-fold for clone C2) (Fig. 3⇓A, lanes 6 and 10) compared with control cells (Fig. 3⇓A, lane 2). Similar results were obtained with the levels of COX-2 protein expression, as in clone B1 (Fig. 3⇓B, lane 6) and in clone C2 (Fig. 3⇓B, lane 10) COX-2 levels were increased by 2- and 4-fold, respectively, compared with the levels observed in control cells (Fig. 3⇓B, lane 2). This significant increase in IFN-γ-induced COX-2 expression in DN PKC-α-overexpressing RAW 264.7 cells suggested that PKC-α negatively modulates IFN-γ-induced COX-2 expression. When macrophages were exposed to a combination of both IFN-γ and LPS, high levels of COX-2 mRNA and protein were induced independently of DN PKC-α overexpression (Fig. 3⇓, A and B, lanes 4, 8, and 12). Thus, DN PKC-α overexpression had little effect on the synergistic effect of LPS and IFN-γ on the induction of COX-2 mRNA accumulation and protein synthesis.

FIGURE 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 3.

Effect of DN PKC-α overexpression on COX-2 expression induced by LPS and IFN-γ. Adherent cells (vector alone, clone B1, and clone C2) were incubated in the absence (lanes 1, 5, and 9) or in the presence of either 100 U/ml IFN-γ (lanes 2, 6, and 10), 100 ng/ml LPS (lanes 3, 7, and 11), or a combination of both (lanes 4, 8, and 12) for 8 h. For priming experiments, cells were first incubated with 100 U/ml IFN-γ for 18 h followed by additional stimulation with IFN-γ, or IFN-γ and LPS. Total RNA was extracted, Northern blot analysis was performed (A), and cell extracts were prepared for Western blot analyses (B) as described in Materials and Methods. RNA integrity and loading were assessed by ethidium bromide staining. Similar results were obtained in at least three separate experiments.

COX-2 expression following a phagocytic stimulation with L. donovani promastigotes

Infection with the intracellular protozoan L. donovani stimulates macrophages to secrete PGE2, possibly by inducing COX-2 expression (11, 12). Thus we determined whether PKC-α was involved in this process by comparing the induction of COX-2 mRNA accumulation and protein synthesis in normal RAW 264.7 cells and in DN PKC-α-overexpressing clones following phagocytosis of L. donovani promastigotes. For priming experiments, cells were incubated for 18 h with 100 U/ml IFN-γ before the addition of either 100 U/ml IFN-γ alone or in combination with L. donovani promastigotes for an additional 8 h. Phagocytic stimulation with L. donovani promastigotes failed to induce COX-2 mRNA accumulation as well as protein synthesis in control RAW 264.7 macrophages (Fig. 4⇓, A and B, lane 3) and in the two DN PKC-α-overexpressing clones (Fig. 4⇓, A and B, lane 7 for clone B1 and lane 11 for clone C2). Priming with IFN-γ had no effect on the induction of COX-2 expression following phagocytosis of L. donovani, as COX-2 mRNA and protein levels induced by IFN-γ alone (Fig. 4⇓, A and B, lane 2 for control cells, lane 6 for clone B1, and lane 10 for clone C2) were similar to those induced by the combination of IFN-γ and L. donovani (Fig. 4⇓, A and B, lane 4 for control cells, lane 8 for clone B1, and lane 12 for clone C2). In naive BMM, L. donovani evaded the induction of COX-2 expression (Fig. 5⇓, A and B, lane 5), whereas priming with IFN-γ led to the induction of COX-2 mRNA and protein synthesis by L. donovani promastigotes in BMM (Fig. 5⇓, A and B, lane 6).

FIGURE 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 4.

Effect of DN PKC-α overexpression on COX-2 expression induced by L. donovani promastigotes. Adherent cells (vector alone, clone B1, and clone C2) were incubated in the absence (lanes 1, 5, and 9) or in the presence of different stimuli, namely, 100 U/ml IFN-γ (lanes 2, 6, and 10), L. donovani promastigotes (lanes 3, 7, and 11), or a combination of both (lanes 4, 8, and 12) for 8 h. For priming experiments, cells were first incubated with 100 U/ml IFN-γ for 18 h followed by additional stimulation with L. donovani and IFN-γ, or IFN-γ alone. Total RNA was extracted, Northern blot analysis was performed (A), and cell extracts were prepared for Western blot analyses (B) as described in Materials and Methods. RNA integrity and loading were assessed by ethidium bromide staining. Similar results were obtained in at least three separate experiments.

FIGURE 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 5.

COX-2 expression in BMM. BMM were incubated in the absence (lane 1) or in the presence of different stimuli (lanes 2–6) for 8 h. For priming experiments, cells were first incubated with 100 U/ml IFN-γ for 18 h followed by 100 ng/ml LPS, L. donovani promastigotes, or 100 U/ml IFN-γ. Total RNA was extracted, Northern blot analysis was performed (A), and cell extracts were prepared for Western blot analyses (B) as described in Materials and Methods. RNA integrity and loading were assessed by ethidium bromide staining. Similar results were obtained in at least three separate experiments.

Effect of DN PKC-α overexpression on PGE2 secretion

We compared the ability of control RAW 264.7 cells and clones B1 and C2 to secrete PGE2 in response to either LPS (10 or 100 ng/ml), 100 U/ml IFN-γ, or L. donovani. As shown in Fig. 6⇓A, in the presence of 10 ng/ml (▧) and 100 ng/ml (▪) LPS, control RAW 264.7 cells as well as DN PKC-α-overexpressing clones B1 and C2 secreted PGE2 in a dose-dependent manner. In contrast to COX-2 mRNA and protein levels, overexpression of DN PKC-α did not affect LPS-induced PGE2 secretion by RAW 264.7 cells. (For 10 ng/ml LPS, p = 0.15 for B1 vs control cells, and p = 0.02 for C2 vs control cells, n = 3. For 100 ng/ml LPS, p = 0.103 for B1 vs control cells, and p = 0.07 for C2 vs control cells, n = 3.) Data obtained with the specific COX-2 inhibitor NS-398 (5 μM) (25) confirmed that COX-2 activation is the major pathway responsible for LPS-stimulated PGE2 secretion (Table I⇓). The observation that valeryl salicylate, a COX-1 inhibitor (26), reduced LPS-induced PGE2 production by 50% suggested a role for COX-1, although it is possible that COX-2 activity was also inhibited at the concentration used (1 mM) (Table I⇓). As shown in Fig. 6⇓B, IFN-γ induced the secretion of minimal PGE2 levels in control RAW 264.7 cells, whereas DN PKC-α-overexpression increased IFN-γ-induced PGE2 secretion by 35-fold by clone B1 (p = 0.005, n = 3) and 70-fold by clone C2 (p = 0.01, n = 3). Collectively, these results indicated that DN PKC-α overexpression had no effect on LPS-induced PGE2 secretion but strongly up-regulated IFN-γ-induced PGE2 secretion. When macrophages were exposed to a combination of IFN-γ and LPS, control RAW 264.7 cells and the DN PKC-α-overexpressing clones B1 and C2 secreted similar PGE2 levels (p = 0.04 for B1 vs control cells, and p = 0.795 for C2 vs control cells, n = 3) (Fig. 6⇓C). As observed for COX-2 mRNA and protein synthesis, L. donovani promastigotes failed to induce PGE2 secretion in control RAW 264.7 cells as well as in DN PKC-α-overexpressing clones B1 and C2 (data not shown).

FIGURE 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
FIGURE 6.

Effect of DN PKC-α on PGE2 secretion. Adherent cells (vector alone, clone B1, and clone C2) were incubated in the absence or presence of either 10 ng/ml or 100 ng/ml LPS (A), 100 U/ml IFN-γ (B), or a combination of 100 U/ml IFN-γ and 100 ng/ml LPS (C) for 8 h. Cells stimulated with IFN-γ were first incubated for 18 h with 100 U/ml IFN-γ. Levels of PGE2 in cell supernatants were determined by ELISA as described in Materials and Methods. Experiments were performed in triplicate and are representative of results obtained in three separate experiments. ∗, p < 0.05; ∗∗, p < 0.01 as compared with IFN-γ-treated control cells.

View this table:
  • View inline
  • View popup
Table I.

Effect of COX and iNOS inhibitors on LPS-induced PGE2 secretion in RAW 264.7 macrophagesa

Discussion

PGs are important regulatory mediators for the maintenance of numerous physiological functions and are synthesized by most mammalian tissues (3, 27). In inflammatory reactions, macrophages are the main producers of large quantities of PGE2 (10, 28). COX-2, the inducible COX isoform, has been identified in activated macrophages and constitutes the key enzyme responsible for the high production of inflammatory PGs such as PGE2 (3, 5, 29). A role for PKC in the regulation of PG production (possibly by regulating COX-2 expression) has been suggested after treatment of macrophages with PKC inhibitors or activators (1, 13). In this study, we investigated the role of PKC-α in the regulation of COX-2 expression in macrophages. To this end, we have stably overexpressed a DN mutant of this isoenzyme in the murine macrophage cell line RAW 264.7 (16). We obtained evidence suggesting that PKC-α activity is important for the modulation of COX-2 expression in macrophages exposed to either LPS or IFN-γ.

Previous studies suggested that PKC is involved in the regulation of COX-2 expression. A role for PKC-α in the regulation of zymosan-induced PGE2 secretion in mouse peritoneal macrophages has been previously proposed based on the selective down-regulation of PKC isoenzymes and on their differential sensitivities to pharmacological inhibitors (17). Recently, it has been reported that overexpression of wild-type PKC-α in mouse epidermis increases phorbol esters-induced expression of specific proinflammatory mediators, including COX-2, suggesting that PKC-α plays a role in cutaneous inflammation (30). Our data obtained with DN PKC-α-overexpressing clones suggest that this isoenzyme is required for COX-2 expression in the RAW 264.7 macrophage cell line. However, the possibility exists that stable overexpression of the DN PKC-α might have affected basal PKC-α activity levels, thereby altering the maintenance of normal cellular functions. A possible consequence of such alterations could be the loss of regulator(s), which could potentially affect signal transduction pathways leading to COX-2 expression. Accordingly, PKC-α would play a secondary role in LPS-induced COX-2 expression. In contrast, our observation that overexpression of wild-type PKC-α increases LPS-induced COX-2 promoter activity is not consistent with this possibility and argues in favor of a direct role for PKC-α (Fig. 2⇑). Thus, our observations further support a role for PKC-α in the modulation of COX-2 expression in macrophages, and hence in the regulation of inflammatory responses.

The mechanism by which PKC-α modulates COX-2 expression remains obscure. One possibility is that PKC-α is required for the activation of specific transcription factors. In this regard, few studies have addressed the identity of the transcription factor(s) regulating COX-2 expression (31, 32, 33). The ubiquitous transcription factor NF-κB, one of the main mediators of LPS responses (34), binds to regulatory sequences within the promoter region (−403 to −395 bp) of both the human and mouse COX-2 genes to regulate COX-2 expression (35). Because LPS-induced NF-κB activation takes place normally in DN PKC-α-overexpressing macrophages (16), it is likely that transcription factor(s) other than NF-κB and required for LPS-induced COX-2 expression may be defective in our DN PKC-α overexpressing clones. Consensus binding sites for NF-IL6 have been identified within the COX-2 promoter region, and recent evidence indicated that this regulatory sequence is responsible for the induction of human COX-2 by LPS, through NF-IL6β (C/EBPδ) (33). More recently, it has been established that although that NF-κB is not required, NF-IL6 is essential for LPS-induced COX-2 gene expression in RAW 264.7 cells (36). Further studies will be required to examine whether a defective activation of NF-IL6 could account for the inhibition of LPS-induced COX-2 expression in the DN PKC-α overexpressing RAW 264.7 macrophages. In this regard, preliminary evidence indicated that DN PKC-α overexpression inhibited LPS-induced NF-IL6 activation in RAW 264.7 cells (F. Chano and A. Descoteaux, unpublished data).

IFN-γ is a pleiotropic cytokine that plays a key role in modulating immune and inflammatory responses (37) and regulates several macrophage functions (38). Previous studies in human macrophages demonstrated that IFN-γ priming is required for the induction of COX-2 expression following stimulation with either IFN-γ or TNF-α. Moreover, IFN-γ, in combination with either LPS or TNF-α, induced a synergistic increase in the accumulation of COX-2 mRNA (24). However, this synergistic effect is not universal, as IFN-γ priming down-regulated COX-2 gene transcription in response to IL-1β but not to LPS in human macrophages (6). Despite these observations, no data exist on the regulation of COX-2 expression by PKC following stimulation with IFN-γ. In contrast to LPS-induced COX-2 expression, we found that levels of COX-2 mRNA were significantly enhanced in DN PKC-α-overexpressing macrophages following a stimulation with IFN-γ. These data suggest that PKC-α negatively modulates COX-2 expression in response to IFN-γ. Two possible mechanisms may account for these results. First, overexpression of DN PKC-α influences the transcriptional activity of the COX-2 promoter, possibly by regulating the activation of IFN-γ-induced transcription factor(s). IFN consensus sequence binding protein (ICSBP), which is primarily expressed in cells of the macrophage and lymphocytic lineages, is a member of the IFN regulatory factor family that binds to a DNA sequence, known as the IFN-stimulated response element (ISRE), which mediates IFN-γ responsiveness for several genes (39, 40). ICSBP mRNA levels become elevated in response to IFN-γ, but not IFN-α/β, in macrophage cell lines and in thioglycollate-elicited peritoneal macrophages (41). Thus, the selectivity of ICSBP for macrophages and other cells of the immune system, coupled with its strong inducibility and long half-life in macrophages, suggests that it could play a critical role in the down-regulation of macrophage activity after activation by IFN-γ (42). Recent studies provided evidence that ICSBP can selectively suppress the expression of IFN-responsive genes (40). Furthermore, induction of ICSBP mRNA by IFN-γ was found previously to be inhibited by PKC inhibitors (41). Considering these observations, it will be of interest to verify the role of ICSBP in IFN-γ-induced COX-2 expression in DN PKC-α overexpressing RAW 264.7 cells in response to IFN-γ. Second, the steady-state levels of COX-2 transcripts are the result of a balance between the rate of gene transcription and the rate of degradation of the mRNA produced. The 3′ untranslated region of COX-2 mRNA contains conserved AUUA repeats also found in other short-lived mRNA species, such as GM-CSF mRNA (20, 43), that are important in determining mRNA stability and translation (44, 45). Whether PKC-α activity negatively regulates the binding of putative cytosolic factors to the 3′ untranslated region of the COX-2 transcripts, and hence influences COX-2 mRNA stability in IFN-γ-stimulated macrophages, is an hypothesis that will deserve further attention.

L. donovani is an obligate intracellular protozoan that resides within mononuclear phagocytes of infected mammals (46). A previous study demonstrated that infection of murine peritoneal macrophages with L. donovani induced specific alterations in COX and lipoxygenase pathways. This response involved selective increase of some metabolites, such as PGE2 (11). Another study in spleen cells indicated an ex vivo evidence for increased COX activity (12). Because LPS- and IFN-γ-induced COX-2 expression are modulated by PKC-α it was of interest to determine whether DN PKC-α overexpression would influence COX-2 expression during phagocytosis of L. donovani promastigotes. However, we failed to detect COX-2 expression in RAW 264.7 cells exposed to L. donovani promastigotes. In contrast to RAW 264.7 cells, IFN-γ treatment of BMM before infection with L. donovani promastigotes allowed the induction of COX-2 expression.

Whereas COX-2 mRNA and protein synthesis were inhibited, LPS-induced PGE2 secretion was normal in DN PKC-α-overexpressing macrophages. A recent study reported that secretion of NO attenuates PGE2 production in response to LPS in RAW 264.7 macrophages (47). Moreover, it was shown that NO suppresses the activity and expression of COX-2 mRNA in LPS-stimulated rat peritoneal macrophages (48). However, data obtained with the iNOS inhibitor l-NMMA (Table I⇑) ruled out the possibility that our data are related to the low levels of NO secreted by LPS-stimulated DN PKC-α-overexpressing clones (16). In contrast, PGE2 secretion was increased in DN PKC-α-overexpressing clones compared with control RAW 264.7 cells in response to IFN-γ.

In summary, we have provided evidence suggesting a role for PKC-α in the modulation of COX-2 expression in macrophages. Further knowledge of the mechanism that regulates COX-2 expression may potentially lead to the development of novel anti-inflammatory therapies.

Acknowledgments

We thank K. Chadee for critical comments and helpful discussions, J. Giroux for helping with calculations, and H. R. Herschman for the COX-2-luciferase reporter construct pTIS10L.

Footnotes

  • ↵1 This work was supported by Grant MT-12933 from the Medical Research Council of Canada and from an establishment grant from the Fonds pour la Formation de Chercheurs et l’Aide à la Recherche du Québec. A.D. is a Medical Research Council Scholar.

  • ↵2 Address correspondence and reprint requests to Dr. Albert Descoteaux, Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, Québec, Canada H7V 1B7. E-mail address: albert.descoteaux{at}iaf.uquebec.ca

  • ↵3 Abbreviations used in this paper: COX, cyclooxygenase; PKC, protein kinase C; BMM, bone marrow derived-macrophage(s); DN, dominant-negative; iNOS, inducible NO synthase; ICSBP, IFN consensus sequence binding protein; l-NMMA, NG-monomethyl-l-arginine monoacetate.

  • Received November 17, 1999.
  • Accepted July 14, 2000.
  • Copyright © 2000 by The American Association of Immunologists

References

  1. ↵
    Burch, R. M.. 1987. Protein kinase C mediates endotoxin and zymosan-induced prostaglandin synthesis. Eur. J. Pharmacol. 142: 431
    OpenUrlCrossRefPubMed
  2. ↵
    Russell, S. W., J. L. Pace. 1984. Both the kind and magnitude of stimulus are important in overcoming the negative regulation of macrophage activation by PGE2. J. Leukocyte Biol. 35: 291
    OpenUrlAbstract
  3. ↵
    Dubois, R. N., S. B. Abramson, L. Crofford, R. A. Gupta, L. S. Simon, L. B. Van De Putte, P. E. Lipsky. 1998. Cyclooxygenase in biology and disease. FASEB J. 12: 1063
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Langenbach, R., S. G. Morham, H. F. Tiano, C. D. Loftin, B. I. Ghanayem, P. C. Chulada, J. F. Mahler, C. A. Lee, E. H. Goulding, K. D. Kluckman. 1995. Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration. Cell 83: 483
    OpenUrlCrossRefPubMed
  5. ↵
    Morham, S. G., R. Langenbach, C. D. Loftin, H. F. Tiano, N. Vouloumanos, J. C. Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford, C. A. Lee. 1995. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 83: 473
    OpenUrlCrossRefPubMed
  6. ↵
    Barrios-Rodiles, M., K. Chadee. 1998. Novel regulation of cyclooxygenase-2 expression and prostaglandin E2 production by IFN-γ in human macrophages. J. Immunol. 161: 2441
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Lee, S. H., E. Soyoola, P. Chanmugam, S. Hart, W. Sun, H. Zhong, S. Liou, D. Simmons, D. Hwang. 1992. Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J. Biol. Chem. 267: 25934
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Bachwich, P. R., S. W. Chensue, J. W. Larrick, S. L. Kunkel. 1986. Tumor necrosis factor stimulates interleukin-1 and prostaglandin E2 production in resting macrophages. Biochem. Biophys. Res. Commun. 136: 94
    OpenUrlCrossRefPubMed
  9. ↵
    Hla, T., K. Neilson. 1992. Human cyclooxygenase-2 cDNA. Proc. Natl. Acad. Sci. USA 89: 7384
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Riese, J., T. Hoff, A. Nordhoff, D. L. DeWitt, K. Resch, V. Kaever. 1994. Transient expression of prostaglandin endoperoxide synthase-2 during mouse macrophage activation. J. Leukocyte Biol. 55: 476
    OpenUrlAbstract
  11. ↵
    Reiner, N. E., C. J. Malemud. 1985. Arachidonic acid metabolism by murine peritoneal macrophages infected with Leishmania donovani: in vitro evidence for parasite-induced alterations in cyclooxygenase and lipoxygenase pathways. J. Immunol. 134: 556
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Reiner, N. E., C. J. Malemud. 1984. Arachidonic acid metabolism in murine leishmaniasis (Donovani): ex-vivo evidence for increased cyclooxygenase and 5-lipoxygenase activity in spleen cells. Cell. Immunol. 88: 501
    OpenUrlCrossRefPubMed
  13. ↵
    Pfannkuche, H. J., V. Kaever, K. Resch. 1986. A possible role of protein kinase C in regulating prostaglandin synthesis of mouse peritoneal macrophages. Biochem. Biophys. Res. Commun. 139: 604
    OpenUrlCrossRefPubMed
  14. ↵
    Newton, A. C.. 1995. Protein kinase C: structure, function, and regulation. J. Biol. Chem. 270: 28495
    OpenUrlFREE Full Text
  15. ↵
    Blobe, G. C., S. Stribling, L. M. Obeid, Y. A. Hannun. 1996. Protein kinase C isoenzymes: regulation and function. Cancer Surv. 27: 213
    OpenUrlPubMed
  16. ↵
    St-Denis, A., F. Chano, P. Tremblay, Y. St-Pierre, A. Descoteaux. 1998. Protein kinase C-α modulates lipopolysaccharide-induced functions in a murine macrophage cell line. J. Biol. Chem. 273: 32787
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Huwiler, A., J. Pfeilschifter. 1993. A role for protein kinase C-α in zymosan-stimulated eicosanoid synthesis in mouse peritoneal macrophages. Eur. J. Biochem. 217: 69
    OpenUrlPubMed
  18. ↵
    Descoteaux, A., G. Matlashewski. 1989. c-fos and tumor necrosis factor gene expression in Leishmania donovani-infected macrophages. Mol. Cell. Biol. 9: 5223
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Descoteaux, A., G. Matlashewski. 1990. Regulation of tumor necrosis factor gene expression and protein synthesis in murine macrophages treated with recombinant tumor necrosis factor. J. Immunol. 145: 846
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Fletcher, B. S., D. A. Kujubu, D. M. Perrin, H. R. Herschman. 1992. Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase. J. Biol. Chem. 267: 4338
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Finkenzeller, G., D. Marme, H. Hug. 1990. Sequence of human protein kinase C α. Nucleic Acids Res. 18: 2183
    OpenUrlFREE Full Text
  22. ↵
    Smith, W. L., R. M. Garavito, D. L. DeWitt. 1996. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J. Biol. Chem. 271: 33157
    OpenUrlFREE Full Text
  23. ↵
    Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, R. D. Schreiber. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67: 227
    OpenUrlCrossRefPubMed
  24. ↵
    Arias-Negrete, S., K. Keller, K. Chadee. 1995. Proinflammatory cytokines regulate cyclooxygenase-2 mRNA expression in human macrophages. Biochem. Biophys. Res. Commun. 208: 582
    OpenUrlCrossRefPubMed
  25. ↵
    Futaki, N., I. Arai, Y. Hamasaka, S. Takahashi, S. Higuchi, S. Otomo. 1993. Selective inhibition of NS-398 on prostanoid production in inflamed tissue in rat carrageenan-air-pouch inflammation. J. Pharm. Pharmacol. 45: 753
    OpenUrlCrossRefPubMed
  26. ↵
    Davidson, M. E., R. J. Lang. 2000. Effects of selective inhibitors of cyclo-oxygenase-1 (COX-1) and cyclo-oxygenase-2 (COX-2) on the spontaneous myogenic contractions in the upper urinary tract of the guinea-pig and rat. Br. J. Pharmacol. 129: 661
    OpenUrlCrossRefPubMed
  27. ↵
    Williams, J. A., E. Shacter. 1997. Regulation of macrophage cytokine production by prostaglandin E2: distinct roles of cyclooxygenase-1 and -2. J. Biol. Chem. 272: 25693
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Phipps, R. P., S. H. Stein, R. L. Roper. 1991. A new view of prostaglandin E regulation of the immune response. Immunol. Today 12: 349
    OpenUrlCrossRefPubMed
  29. ↵
    Pennisi, E.. 1998. Building a better aspirin. Science 280: 1191
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Wang, H. Q., R. C. Smart. 1999. Overexpression of protein kinase C-α in the epidermis of transgenic mice results in striking alterations in phorbol ester-induced inflammation and COX-2, MIP-2 and TNF-α expression but not tumor promotion. J. Cell Sci. 112: 3497
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Bauer, M. K., K. Lieb, K. Schulze-Osthoff, M. Berger, P. J. Gebicke-Haerter, J. Bauer, B. L. Fiebich. 1997. Expression and regulation of cyclooxygenase-2 in rat microglia. Eur. J. Biochem. 243: 726
    OpenUrlPubMed
  32. ↵
    D’Acquisto, F., T. Iuvone, L. Rombola, L. Sautebin, M. Di Rosa, R. Carnuccio. 1997. Involvement of NF-κB in the regulation of cyclooxygenase-2 protein expression in LPS-stimulated J774 macrophages. FEBS Lett. 418: 175
    OpenUrlCrossRefPubMed
  33. ↵
    Kim, Y., S. M. Fischer. 1998. Transcriptional regulation of cyclooxygenase-2 in mouse skin carcinoma cells: regulatory role of CCAAT/enhancer-binding proteins in the differential expression of cyclooxygenase-2 in normal and neoplastic tissues. J. Biol. Chem. 273: 27686
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Sweet, M. J., D. A. Hume. 1996. Endotoxin signal transduction in macrophages. J. Leukocyte Biol. 60: 8
    OpenUrlAbstract
  35. ↵
    Yamamoto, K., T. Arakawa, N. Ueda, S. Yamamoto. 1995. Transcriptional roles of nuclear factor κ B and nuclear factor-interleukin-6 in the tumor necrosis factor α-dependent induction of cyclooxygenase-2 in MC3T3–E1 cells. J. Biol. Chem. 270: 31315
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Wadleigh, D. J., S. T. Reddy, E. Kopp, S. Ghosh, H. R. Herschman. 2000. Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages. J. Biol. Chem. 275: 6259
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Young, H. A., K. J. Hardy. 1995. Role of interferon-γ in immune cell regulation. J. Leukocyte Biol. 58: 373
    OpenUrlAbstract
  38. ↵
    Adams, D. O., T. A. Hamilton. 1987. Molecular transductional mechanisms by which IFN γ and other signals regulate macrophage development. Immunol. Rev. 97: 5
    OpenUrlCrossRefPubMed
  39. ↵
    Friedman, R. L., G. R. Stark. 1985. α-Interferon-induced transcription of HLA and metallothionein genes containing homologous upstream sequences. Nature 314: 637
    OpenUrlCrossRefPubMed
  40. ↵
    Kantakamalakul, W., A. D. Politis, S. Marecki, T. Sullivan, K. Ozato, M. J. Fenton, S. N. Vogel. 1999. Regulation of IFN consensus sequence binding protein expression in murine macrophages. J. Immunol. 162: 7417
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Politis, A. D., J. Sivo, P. H. Driggers, K. Ozato, S. N. Vogel. 1992. Modulation of interferon consensus sequence binding protein mRNA in murine peritoneal macrophages. Induction by IFN-γ and down-regulation by IFN-α, dexamethasone, and protein kinase inhibitors. J. Immunol. 148: 801
    OpenUrlAbstract
  42. ↵
    Politis, A. D., K. Ozato, J. E. Coligan, S. N. Vogel. 1994. Regulation of IFN-γ-induced nuclear expression of IFN consensus sequence binding protein in murine peritoneal macrophages. J. Immunol. 152: 2270
    OpenUrlAbstract
  43. ↵
    Shaw, G., R. Kamen. 1986. A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46: 659
    OpenUrlCrossRefPubMed
  44. ↵
    Hel, Z., S. Di Marco, D. Radzioch. 1998. Characterization of the RNA binding proteins forming complexes with a novel putative regulatory region in the 3®-UTR of TNF-α mRNA. Nucleic Acids Res. 26: 2803
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Gueydan, C., L. Houzet, A. Marchant, A. Sels, G. Huez, V. Kruys. 1996. Engagement of tumor necrosis factor mRNA by an endotoxin-inducible cytoplasmic protein. Mol. Med. 2: 479
    OpenUrlPubMed
  46. ↵
    Liew, F. Y., C. A. O’Donnell. 1993. Immunology of leishmaniasis. Adv. Parasitol. 32: 161
    OpenUrlCrossRefPubMed
  47. ↵
    Patel, R., M. G. Attur, M. Dave, S. B. Abramson, A. R. Amin. 1999. Regulation of cytosolic COX-2 and prostaglandin E2 production by nitric oxide in activated murine macrophages. J. Immunol. 162: 4191
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Habib, A., C. Bernard, M. Lebret, C. Creminon, B. Esposito, A. Tedgui, J. Maclouf. 1997. Regulation of the expression of cyclooxygenase-2 by nitric oxide in rat peritoneal macrophages. J. Immunol. 158: 3845
    OpenUrlAbstract
PreviousNext
Back to top

In this issue

The Journal of Immunology: 165 (7)
The Journal of Immunology
Vol. 165, Issue 7
1 Oct 2000
  • Table of Contents
  • About the Cover
Print
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for your interest in spreading the word about The Journal of Immunology.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Cyclooxygenase-2 Expression in Macrophages: Modulation by Protein Kinase C-α
(Your Name) has forwarded a page to you from The Journal of Immunology
(Your Name) thought you would like to see this page from the The Journal of Immunology web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Cyclooxygenase-2 Expression in Macrophages: Modulation by Protein Kinase C-α
Mélanie Giroux, Albert Descoteaux
The Journal of Immunology October 1, 2000, 165 (7) 3985-3991; DOI: 10.4049/jimmunol.165.7.3985

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Cyclooxygenase-2 Expression in Macrophages: Modulation by Protein Kinase C-α
Mélanie Giroux, Albert Descoteaux
The Journal of Immunology October 1, 2000, 165 (7) 3985-3991; DOI: 10.4049/jimmunol.165.7.3985
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like

Jump to section

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • Suppression of inflammation in collagen-induced arthritis by administration of recombinant Fcγ receptors (54.17)
  • The immunological ménage à trois promotes inflammation in Type 2 diabetes. (54.14)
  • Identification of novel small molecule inducers of endothelium-driven innate immunity (54.10)
Show more Inflammation

Similar Articles

Navigate

  • Home
  • Current Issue
  • Next in The JI
  • Archive
  • Brief Reviews
  • Pillars of Immunology
  • Translating Immunology

For Authors

  • Submit a Manuscript
  • Instructions for Authors
  • About the Journal
  • Journal Policies
  • Editors

General Information

  • Advertisers
  • Subscribers
  • Rights and Permissions
  • Accessibility Statement
  • Public Access
  • Privacy Policy
  • Disclaimer

Journal Services

  • Email Alerts
  • RSS Feeds
  • ImmunoCasts
  • Twitter

Copyright © 2021 by The American Association of Immunologists, Inc.

Print ISSN 0022-1767        Online ISSN 1550-6606