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The Journal of Immunology, 2008, 180, 6903 -6910
Copyright © 2008 by The American Association of Immunologists, Inc.
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Agonists of Proteinase-Activated Receptor-2 Enhance IFN-{gamma}-Inducible Effects on Human Monocytes: Role in Influenza A Infection1

Micha Feld2,*, Victoria M. Shpacovitch2,3,*, Christina Ehrhardt{dagger}, Claus Kerkhoff{ddagger}, Morley D. Hollenberg§, Nathalie Vergnolle§, Stephan Ludwig{dagger} and Martin Steinhoff*

* Department of Dermatology, Interdisziplinäres Zentrum für Klinische Forschung Münster, and Boltzmann Institute for Immunobiology of the Skin, {dagger} Department of Molecular Virology, Zentrum für Molekularbiologie der Entzündung, and {ddagger} Department of Experimental Dermatology, University of Münster, Münster, Germany; and § Canadian Institutes of Health Research Proteinases and Inflammation Network, Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Proteinase-activated receptor-2 (PAR2) is expressed by different types of human leukocytes and involved in the development of inflammatory and infectious diseases. However, its precise role in the regulation of human monocyte and macrophage function during viral infection remains unclear. Also, the ability of PAR2 agonists to enhance the effects induced by immune mediators during infection or inflammation is still poorly investigated. Therefore, we investigated the ability of a PAR2 agonist to enhance IFN-{gamma}-induced suppression of influenza A virus replication in human monocytes. We found that this effect correlates with an increased abundance of I{kappa}B{alpha} after costimulation of cells with PAR2 agonist and IFN-{gamma}. Remarkably, coapplication of PAR2 agonist and IFN-{gamma} also enhances the effects of IFN-{gamma} on IFN-{gamma}-inducible protein 10 kDa release, and CD64 and {alpha}Vβ3 surface expression by human monocytes. Together, these findings indicate a potentially protective role of PAR2 activation during the progression of influenza A virus infection. This effect could be associated with the ability of PAR2 agonists to enhance IFN-{gamma}-induced protective effects on human monocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Proteinase-activated receptor 2 (PAR2)4 belongs to a new subfamily of G-protein coupled receptors, activated by serine proteases via cleavage of the extracellular N-terminal sequence. Subsequently, the new amino-terminal sequence of "tethered ligand," unmasked upon enzymatic cleavage, interacts with the extracellular domain of the receptor and activates it (1). Naturally, PAR2 is activated by trypsins, kallikreins, coagulation enzymes (factors FVIIa and FXa), proteases derived from immune cells (tryptase and elastase), or pathogens (gingipains and house dust mite allergens) (reviewed in Refs. 2, 3). This receptor is expressed on human leukocytes such as neutrophils, monocytes/macrophages, and mast cells (reviewed in Ref. 3). Accumulating evidence indicates a role of PAR2 in the development of various inflammatory diseases (reviewed in Refs. 1, 2). The impact of PAR2 activation on inflammation can be variable, in some instances leading to proinflammatory effects (4, 5), and in other settings to an anti-inflammatory protective effect (6, 7).

In mice, PAR2 expression in airways is enhanced upon infection with influenza A virus, pointing to the involvement of this receptor in the pathogenesis of viral disease (8). Moreover, PAR2-expressing mononuclear cells were found to infiltrate the infected airway tissues during influenza viral infection (8). Nonetheless, the precise role of PAR2 in regulating monocyte function during influenza A virus infection has not been evaluated in any depth.

Upon infection, human monocytes/macrophages release immunoactive mediators (for example, IL-18 and IFN{alpha}/β) directing infiltrating cells (monocytes and lymphocytes) to the site of infection and inducing anti-viral activities (9, 10). Macrophage-derived IFN{alpha}/β and IL-18 regulate production of IFN-{gamma} by T and NK cells (11). This cytokine, in turn, enhances monocyte/macrophage activation acting on production of anti-viral mediators and expression of cell surface molecules involved in ingestion of opsonized viral particles and in process of monocyte transmigration (Fc{gamma}RI and {alpha}Vβ3, respectively) (10, 12, 13).

IFNs, originally discovered as agents that interfere with viral replication, are now well-known as immune mediators with a wide range of functions. IFNs amplify anti-viral mechanisms affecting cleavage and "editing" of viral RNA, inhibition of protein synthesis, and transcription (reviewed in Ref. 14). Although the immunomodulatory activity of IFN-{gamma} is often considered as its primary function, this cytokine is also very important for establishing an effective anti-viral defense (15). Because of its recognized ability to regulate an inflammatory-protective response, we wondered whether PAR2 might synergize with the actions of IFN-{gamma}.

In the present study, we hypothesized that PAR2 agonists may act along with IFN-{gamma} to enhance the suppression of viral replication and to increase the production of an inflammatory cytokine like IFN-{gamma}-inducible protein 10 kDa (IP-10). We also hypothesized that PAR2 agonist would enhance IFN-{gamma}-induced up-regulation of the expression on monocyte cell surface of {alpha}Vβ3 integrin and CD64 (Fc{gamma}RI), the molecules that participate in migration of monocytes toward opsonized virus particles. To test these hypotheses, we measured the viral yield of influenza A-infected monocytes treated with a PAR2 agonist and IFN-{gamma} either alone or in combination, and we measured the production of IP-10 by the cells. We also determined the cell surface expression of CD64 (Fc{gamma}RI) and {alpha}Vβ3 integrin in monocytes treated with the same two agonists either alone or in combination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

Human rIFN-{gamma} was purchased from TebuBio. Trypsin was purchased from Sigma-Aldrich. Human PAR2 activating peptide with the sequence trans-cinnamoyl-LIGRLO-NH2 (cAP) and reverse peptide with sequence trans-cinnamoyl-OLRGIL-NH2 (cRP) (from Dr. McMaster, University of Calgary, Calgary, Canada) were used as described previously (16). The following mAbs were used: unconjugated mouse anti-human PAR2 (clone SAM-11) (Santa Cruz Biotechnology), mouse anti-human CD64 (Dako), mouse anti-human {alpha}Vβ3 (Chemicon International), mouse anti-human I{kappa}B{alpha} (Imgenex), as well as mouse anti-human β-Actin (Sigma-Aldrich). HRP-conjugated sheep anti-mouse Ab was obtained from Amersham Biosciences, and PE-conjugated goat anti-mouse Ab was obtained from Jackson ImmunoResearch Laboratories. Cell culture reagents were from BioWhittaker, PromoCell, and Life Technologies. fMLP was purchased from Sigma-Aldrich. Fura-2 acetoxymethyl ester was from Invitrogen. Avian influenza virus A/FPV/Bratislava/79 (H7N7) (FPV) was taken from the depository of the Institute of Molecular Virology, Münster, Germany.

Isolation and culture of human monocytes

Blood for in vitro experiments with human monocytes was obtained from healthy adult volunteers in buffy-coats (Deutsches Rotes Kreuz). Monocytes were isolated by Biocoll (Biochrom) density gradient centrifugation and subsequent negative selection by using magnetic cell sorting and Monocyte Isolation kit II (depletion method) according to the manufacturer’s instructions (Miltenyi Biotech). Isolated monocytes were cultured in RPMI 1640 medium supplemented with 0.9% FCS, 1% penicillin/streptomycin, 2 mM L-glutamine, and 1% nonessential amino acids. Monocytes were cultured at a concentration of 1 x 106 cells per 1.5 ml of medium. After isolation cells were equilibrated (37°C, 5% CO2) for a two hour period to allow for recovery before use in the experiments. IFN-{gamma} was used at a concentration of 200 U/ml; trypsin was used at a concentration of 5 x 10–8 M, which is known to be efficient for human granulocyte stimulation (16); PAR2-tc-activating peptide (PAR2-cAP) was used at concentrations of 1 x 10–5 M, 5 x 10–5 M, 1 x 10–4 M, and 2 x 10–4 M as described in the Results section and figure legends. The corresponding reverse peptide with the reverse-sequence (PAR2-cRP) was used at concentration of 1 x 10–4 M and served as a negative control.

Measurement of cytosolic calcium levels

PAR2 signaling was assessed by measuring cAP-induced Ca2+ mobilization. Monocytes were washed, resuspended in HEPES-buffered salt solution (140 mM NaCl, 3 mM KCl, 0.4 mM Na2HPO4, 10 mM HEPES, 5 mM glucose, 1 mM MgCl2, and 0.8 mM CaCl2 (pH 7.4)), and incubated with 3.5 µM fura-2 acetoxymethyl. Cells were washed twice and resuspended in prewarmed buffer solution. One million cells were transferred to a microcuvette, and the fluorescence of sample was measured at 340 and 380 nm excitation and 510 nm emission in a FluoroMaxx spectrophotometer (Yobin Yvon). The ratio of the fluorescence at the two excitation wavelengths, which is proportional to [Ca2+]i, was calculated.

SDS-PAGE and immunoblot analysis

Whole cell lysates (1 x 106 cells per lane) were separated by SDS-PAGE and blotted onto nitrocellulose membranes (Amersham Biosciences). Membranes were immunostained with either mouse anti-human I{kappa}B{alpha} (1 µg/ml) or mouse anti-human β-Actin (1/2000), and subsequently with sheep anti-mouse HRP (1/3000). Proteins labeled with Ab complexes were visualized using the SuperSignal West Pico ECL detection kit (Pierce).

Virus infections and determination of virus titers in plaque assays

Infection experiments were performed according to the following schemes. In the first scheme of experiments, human monocytes were pretreated for 2 h with PAR2-cAP, PAR2-cRP, IFN-{gamma}, or their combination, and also further stimulated with these substances subsequent to infection with influenza A virus. In the second experimental scheme, human monocytes were only pretreated for 2 h with specified concentrations of trypsin, PAR2-cAP, PAR2-cRP, IFN-{gamma}, or their combination. Further application of substances during virus amplification period was not performed in these experiments.

After the 2 h prestimulation, medium was removed. Cells were washed and were subsequently incubated with influenza virus A/FPV/Bratislava/79 (FPV) at a multiplicity of infection (MOI) of 0.05 diluted in PBS containing 0.01% CaCl2, 0.01% MgCl2, and 0.2% BSA. Incubation was performed for 30 min at 37°C and 5% CO2. Inoculum was aspirated, and monocytes were cultured with or without stimulation in RPMI 1640 supplemented with 2 mM L-glutamine, 1% non essential amino acids, 1% penicillin and streptomycin, 0.2% BSA, 0.01% CaCl2, and 0.01% MgCl2 (here and further "medium for infection"). In the case of stimulated samples, the RPMI 1640 additionally contained PAR2-cAP or PAR2-cRP, IFN-{gamma}, or their combination. Aliquots (400 µl) of the medium were collected at 20 h after infection. Viral replication was determined by a standard plaque assay on confluent Madin-Darby canine kidney cells.

FACS of CD64, {alpha}Vβ3, and PAR2 cell surface expression on human monocytes

For one-color FACS analysis, 1 x 106 monocytes were used. Noninfected monocytes were stimulated with PAR2-cAP, PAR2-cRP (1 x 10–4 M), and/or IFN-{gamma} (200 U/ml). For influenza A infection, monocytes were cultured in RPMI 1640 "medium for infection." Subsequently, cells were incubated with FPV at a MOI of 0.05 for 4, 8, and 12 h. To estimate cell surface expression of investigated molecules appropriate primary Abs were applied to cells in the ratio 1 µg per 1 x 106 cells in 100 µl. For further staining, PE-conjugated goat anti-mouse Ab (1/50) was used. Monocytes incubated with secondary Ab alone served as a negative control. At least 20,000 cells were analyzed with the FACScalibur and Cell Quest Pro software (BD Biosciences).

ELISA and chemokine arrays

To generate supernatants from noninfected monocytes, 1 x 106 cells were treated with the indicated stimulus for 2 or 16 h, respectively. Supernatants from FPV-infected monocytes were prepared as described above; however, monocytes were pretreated with PAR2-cAP, PAR2-cRP (each 10–4 M), IFN-{gamma} (200 U/ml), or their combination during 16 h. For measurement of cytokine concentration in the culture supernatants, CXCL10/IP-10 ELISA kits from R&D Systems were used according to the manufacturer’s instructions. Screening for different acute phase proteins, cytokines, and chemokines in culture medium was performed with a Proteome profiler array (Panel A Array kit) from R&D Systems (cat. no. ARY005). The array allows to detect the following proteins: C5a, CD40L, G-CSF, GM-CSF, GRO{alpha}, I-309, sICAM-1, IFN-{gamma}, IL-1{alpha}, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-16, IL-17, IL-17E, IL-23, IL-27, IL-32{alpha}, IP-10, I-TAC, MCP-1, MIF, MIP-1{alpha}, MIP-1β, PAI-1, RANTES, SDF-1, TNF-{alpha}, and sTREM-1.

Statistical analysis

Results are expressed as mean ± SEM. At least three independent experiments have been performed (n > 3). Statistical evaluation was performed by paired t test (two tails), sign test (only for plaque assay data as an additional statistical test), or Wilcoxon test. Significance was assigned where p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PAR2-cAP induces [Ca2+]i increase in purified human monocytes

Previously, it was demonstrated that cAP induces an increase in [Ca2+]i in human neutrophils (16). Human primary monocytes are known to express PAR2 mRNA (17, 18), and, in our study, we wanted to confirm that cAP is able to activate PAR2 expressed by human monocytes. Application of cAP at a concentration of 10–4 M induced an increase in intracellular Ca2+ (Fig. 1, A and B). In contrast, treatment of human monocytes with the PAR2-inactive reverse-sequence peptide cRP (10–4 M) failed to cause a response (Fig. 1, A and B). Thus, human monocytes efficiently respond to PAR2-cAP with a specific [Ca2+]i increase.


Figure 1
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  		FIGURE 1. Human monocyte [Ca2+]i response to PAR2-cAP or PAR2-cRP stimulation. Human monocytes were loaded with fura-2AM. Cells were stimulated with 10–4 M PAR2-cAP or 10–4 M PAR2-cRP (served as negative control). Stimulation with 10 nM fMLP was used to verify the viability and responsiveness of monocytes after isolation. Black arrow marks the time point when stimuli were added. A, PAR2-cAP (light gray line on the graph) application induced a significant calcium signal. PAR2-cRP treatment did not result in any changes of [Ca2+]i (gray line on the graph). fMLP stimulation confirmed the viability and responsiveness of monocytes (black line on the graph). B, The difference between the mean of basal [Ca2+]i level and the mean of peak [Ca2+]i ± SEM presented on the graph. Four independent experiments were performed.

 
Effects of PAR2-cAP and IFN-{gamma} on viral replication in isolated human monocytes

In murine epithelial cells, the expression of PAR1 and PAR2 was enhanced during influenza A virus infection (8). Furthermore, IFN-{gamma} expression increases during influenza viral infection of human monocytes (19). However, a precise role of PAR2 activation for viral replication and the ability of PAR2 agonist to enhance IFN-{gamma}-induced protective effects remained unexplored. In the first series of experiments, we pretreated cells for 2 h with PAR2-cAP, IFN-{gamma}, or both before infection with influenza A virus, and also applied these stimuli or their combination during virus replication period (see the description of the first experimental scheme in Materials and Methods for details). In comparison with untreated monocytes, treatment with either the PAR2-cAP (at concentration of 1 x 10–4 M) or IFN-{gamma} (200 U/ml) alone decreased progeny virus titers by ~73 ± 3 and 70 ± 9%, respectively. The combined treatment with the same concentrations of IFN-{gamma} and PAR2-cAP together enhanced the IFN-{gamma}-induced effect and resulted in greater decrease the number of viral particles (92 ± 2% reduction the number of viral particles as compared with untreated cells) (Fig. 2A). In contrast, the control PAR2-cRP (1 x 10–4 M) did not exert any effect on viral replication when it was applied either alone or in combination with IFN-{gamma} (Fig. 2A). To reveal the dose-dependence of the observed cAP effect, we performed a series of experiments with lower (1 x 10–5 M and 5 x 10–5 M) and higher (2 x 10–4 M) concentrations of PAR2-cAP (Fig. 2B). At concentrations 1 x 10–5 M or 5 x 10–5 M, PAR2-cAP did not significantly affect influenza A virus replication and also was not able to enhance IFN-{gamma}-induced anti-viral effect (Fig. 2B). The higher concentration of cAP (2 x 10–4 M) was not more beneficial and protective against viral replication than the concentration used in the first series of experiments (1 x 10–4 M) (compare Fig. 2, A and B). Compared with untreated cells, application of PAR2-cAP at a concentration of 2 x 10–4 M decreased virus titers by 48 ± 14%, whereas combined treatment of 2 x 10–4 M cAP together with IFN-{gamma} reduced virus replication by 93 ± 0.9% (Fig. 2B). Thus, we confirmed that viral-suppressing activity PAR2-cAP is reached at concentration 10–4 M and higher concentrations are not more beneficial. Classical enzymatic PAR2 agonists (trypsin and tryptase) are known to promote directly influenza A virus replication due to their ability to cleave viral hemagglutinin and induce its maturation (20, 21). This fact limited the use of proteases in the first series of experiments, in which both pretreatment and stimuli application during virus amplification period were used. Therefore, we performed an additional series of experiments based only on prestimulation of monocytes with trypsin (5 x 10–8 M), PAR2-cAP (1 x 10–4 M), IFN-{gamma} (200 U/ml), or their combination, without application of these stimuli during viral amplification period (Fig. 2C). However, only the pretreatment of human monocytes with either trypsin or PAR2-cAP was not protective against influenza A virus replication. Moreover, combined application of trypsin or PAR2-cAP along with IFN-{gamma} for 2 h before virus infection did not enhance IFN-{gamma}-induced suppression of influenza A virus replication (Fig. 2C). Thus, PAR2 agonist pretreatment by itself (without agonist application during viral replication) could not efficiently protect human monocytes against viral replication.


Figure 2
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  		FIGURE 2. Determination of influenza A (FPV) virus titers in cell culture supernatants generated by infected human monocytes treated with PAR2-cAP, PAR2-cRP, IFN-{gamma}, or their combination. For the determination, the standard plaque assay method was used (see Materials and Methods section for details). Monocytes were pretreated for 2 h with 10–5, 5 x 10–5, 10–4, or 2 x 10–4 M of cAP; 10–4 M cRP; 200 U/ml IFN-{gamma}; 5 x 10–8 M trypsin; or their combination, and, after pretreatment, infected with 0.05 MOI of FPV. Further, cells were cultured in the presence (A and B) or absence (C) of the stimuli or their combination. The number of plaques formed in a plaque assay after application of supernatants generated by untreated monocytes was considered as 100%. This level of plaque formation served as relative control. Results are presented as an average number of plaques formed after application of monocyte-derived supernatants in standard plaque assay ± SEM. Paired (two-tails) t test was applied. The data were also double-checked by sign test (because non-normal distribution of the data was suspected). Both tests have shown good agreement in the significance. For t test: #, *, p < 0.05; **, p < 0.01; and ***, p < 0.005. The symbol * marks the significance as compared with control and symbol # as compared with IFN-{gamma} sample. A, The results of plaque assay experiments performed with PAR2-cAP, PAR2-cRP (conc. 10–4 M), IFN-{gamma}, or their combination. PAR2 agonist is able to reduce viral replication, when applied alone, and, moreover, to enhance influenza A suppressing activity of IFN-{gamma}. B, Dose-response experiments with different (10–5, 5 x 10–5, or 2 x 10–4 M) concentrations of PAR2 agonist confirm that viral-suppressing activity PAR2-cAP is reached at concentration 10–4 M, and higher concentrations are not more beneficial as compared with this one. C, PAR2 agonist (trypsin 5 x 10–8 M or cAP 10–4 M) pretreatment of monocytes, without further stimuli application during viral replication, is not sufficient for protection of cells against influenza A virus replication. Five independent experiments were performed.

 
Simultaneous application of PAR2-cAP and IFN-{gamma} reduces I{kappa}B{alpha} degradation in human monocytes

Recently, active NF-{kappa}B signaling has been demonstrated to be a prerequisite for efficient influenza A virus infection of human cells (22, 23). The inhibitory subunit, I{kappa}B{alpha}, maintains NF-{kappa}B in an inactive state and is degraded in the course of NF-{kappa}B activation. Therefore, we studied whether PAR2-cAP (1 x 10–4 M) and IFN-{gamma} (200 U/ml) might have an effect on I{kappa}B{alpha} levels in purified monocytes. Activation of cells with the PAR2 agonist had a bi-phasic impact on the abundance of I{kappa}B{alpha}, leading to a rapid early decrease in levels at 15 min and an increased level at 30 min that returned to basal levels at ~2 h after PAR2 agonist application. In contrast, treatment with IFN-{gamma} had little effect on the levels of I{kappa}B{alpha} at 15 and 30 min (Fig. 3, A and B) and caused only an increase in levels at 2 h (Fig. 3C). However, in the presence of both PAR2-cAP and IFN-{gamma} the level of I{kappa}B{alpha} was increased above baseline at all time points (Fig. 3, A–C).


Figure 3
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  		FIGURE 3. Influence of PAR2 and IFN-{gamma} stimulation on I{kappa}B{alpha} degradation. Primary noninfected monocytes were stimulated with 10–4 M cAP, 200 U/ml IFN-{gamma}, or both for 15 min (A), 30 min (B), and 2 h (C). Subsequently, cells were lysed and immunoblotted to reveal the level of I{kappa}B{alpha} abundance. β-actin was used to check the equal loading of samples. As compared with untreated cells, coapplication of PAR2-cAP and IFN-{gamma} reduces intracellular I{kappa}B{alpha} degradation in all investigated time points. Four independent experiments were performed.

 
PAR2-cAP stimulation enhanced IFN-{gamma}-induced IP-10 release from influenza A-infected as well as noninfected human monocytes

We also investigated whether PAR2 agonist application affects IFN-{gamma}-induced changes of chemokine production in monocytes. Enhanced production of IP-10 after monocyte stimulation with IFN-{gamma} is known to attract T lymphocytes and, subsequently, to modulate adaptive immune responses (24). To study the effects of PAR2-cAP (10–4 M), PAR2-cRP (10–4 M), and IFN-{gamma} (200 U/ml) application on chemokine release by human monocytes, we treated cells for 2 or 16 h with the indicated stimulus alone or in combination. Collected cell culture supernatants were further used for chemokine arrays and ELISAs. We were not able to detect any significant difference in IP-10 secretion by human monocytes after stimuli application during 2 h (data not shown). However at 16 h after treatment, we demonstrated that coapplication of IFN-{gamma} and PAR2-cAP enhances the increase of IP-10 release induced by IFN-{gamma} alone in noninfected monocytes (Fig. 4A). Untreated and PAR2-cAP-treated noninfected monocytes did not release IP-10 above the threshold level detectable by chemokine array (Fig. 4A). Using ELISA we performed quantitative analysis of IP-10 release in noninfected as well as infected monocytes. The level of IP-10 release in influenza A virus-infected monocytes, without application of any other stimuli, was 149 ± 60 pg/ml (Fig. 4B). Untreated, noninfected monocytes released IP-10 in a concentration of 9.8 ± 4.3 pg/ml (Fig. 4C). PAR2-cAP alone just slightly enhanced IP-10 release in influenza A virus-infected monocytes (Fig. 4B). However, the effect of PAR2-cAP on IP-10 release by noninfected cells was not significant (Fig. 4C). Coapplication of IFN-{gamma} and PAR2-cAP in influenza A virus-infected monocytes enhanced the IFN-{gamma}-induced effect at ~2-fold (Fig. 4B, right). In noninfected cells, costimulation with PAR2-cAP increased IFN-{gamma}-induced effects ~10-fold (Fig. 4C, right). In contrast, application of PAR2-cRP alone as well as in combination with IFN-{gamma} did not result in any significant changes of IP-10 production by human monocytes (Fig. 4, B and C) (data not shown).


Figure 4
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  		FIGURE 4. Analysis of the changes of IP-10 release by influenza A virus-infected as well as noninfected human monocytes after PAR2-cAP and/or IFN-{gamma} stimulation. Purified monocytes were stimulated with 10–4 M cAP, 200 U/ml IFN-{gamma}, or both for 16 h prior to the measurement of the release of various chemokines. A, Human cytokine protein array (R&D Systems) was applied for qualitative analysis the changes of cytokine and chemokine secretion by noninfected monocytes after stimulation with PAR2-cAP, IFN-{gamma}, or both. Protein array was performed with collected medium according to the manufacturer’s instructions. Presented numbers on "control" and "cAP" membranes to the right of dot pairs mark the following targets: "1" positive controls; "2" IP-10; "3" IL-1ra; "4" IL-16; "5" GRO{alpha}; "6" MIF; "7" IL-8; and "8" PAI-1. The targets have the same positions on all presented membranes. IP-10 secretion by noninfected monocytes increased after IFN-{gamma} stimulation. This effect of IFN-{gamma} was enhanced by simultaneous application of PAR2-cAP. The membrane one of three independent arrays is presented. The levels of IP-10 secreted by influenza virus-infected (B) and noninfected (C) human monocytes were quantified using ELISA (R&D Systems). In the left part of B and C, results are presented as average values of stimuli effects at IP-10 released by monocytes as compared with unstimulated cells ± SEM. Wilcoxon test was applied: *, p < 0.05; **, p < 0.01; and ***, p < 0.005 as compared with untreated cells. In the right part of B and C, the difference in average effects between samples stimulated by IFN-{gamma} alone and in combination with PAR2-cAP was analyzed. Wilcoxon test was applied: ##, p < 0.01 as compared with IFN-{gamma} sample. In the case of infected as well as noninfected monocytes, the coapplication of PAR2-cAP and IFN-{gamma} significantly enhanced the IFN-{gamma}-induced IP-10 secretion. The average value of IP-10 released by influenza A virus-infected, untreated cells was 149 ± 60 pg/ml. Noninfected, untreated monocytes secreted IP-10 at level 9.8 ± 4.3 pg/ml. Three independent experiments were performed for infected as well as noninfected monocytes.

 
Stimulation with IFN-{gamma} enhances PAR2 cell surface expression on human noninfected monocytes

Nystedt and colleagues (25) reported that stimulation of human endothelial cells with TNF-{alpha} or IL-1{alpha} elevated the expression of PAR2 mRNA in these cells. IFN-{gamma} could modulate the level of certain proinflammatory cytokines (TNF-{alpha} and IL-1{alpha}) (26). However, a regulatory role of IFN-{gamma} on PAR2 expression levels is unknown. In this study, we report that the stimulation of isolated noninfected human monocytes with IFN-{gamma} enhances PAR2 cell surface expression in these cells. The display of PAR2 at monocyte cell surface was enhanced at 4 and 8 h after exposure to IFN-{gamma} (the mean fluorescence intensity (MFI) increases at 63 ± 21 and 94 ± 12%, respectively) (Fig. 5, A and B).


Figure 5
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  		FIGURE 5. Analysis of the changes of PAR2 expression on cell surface of human monocytes stimulated with IFN-{gamma} or infected with influenza A (FPV). The PAR2 cell surface expression was assessed by flow cytometry. A and B, Isolated human monocytes were cultured in the presence of IFN-{gamma} (200 U/ml) during 4 (A) or 8 h (B). Monocytes cultured in the absence of IFN-{gamma} served as control. Histograms represent peak overlays between unstimulated control cells (gray shaded peak) and IFN-{gamma} stimulated monocytes (black line peak). IFN-{gamma} enhances PAR2 expression on the cell surface of noninfected human monocytes reaching the maximum efficiency at 8 h after stimulation. C, Human monocytes were infected with FPV (0.05 MOI) during 4, 8, or 12 h. Noninfected monocytes served as control. The average number of PAR2 positive cells as compared with noninfected control ± SEM presented at graph in percents. t test: **, p < 0.01; as compared with noninfected cells. FPV results in a significant down-regulation of PAR2 cell surface expression on monocytes only after 12-h infection. Five independent experiments were performed.

 
PAR2 cell surface expression on human monocytes decreases during influenza A infection

To investigate whether influenza A infection affects cell surface expression of PAR2 on human monocytes, we assessed time-dependent changes of PAR2 display after virus infection of these cells (Fig. 5C). At 4 and 8 h after infection, PAR2 expression did not change significantly (Fig. 5C). However, influenza A virus infection reduced PAR2 display on monocytes at 12 h after infection (the number of PAR2 positive cells decreased at 38 ± 13%) (Fig. 5C). Among noninfected human monocytes, 90 ± 3% were PAR2 positive cells.

Coapplication of PAR2-cAP and IFN-{gamma} enhances IFN-{gamma}-induced changes of CD64 (Fc{gamma}RI) and {alpha}Vβ3 integrin expression on the cell surface of human noninfected monocytes

CD64 is known to play an important role in opsonized phagocytosis during influenza A virus infection (12). The {alpha}Vβ3 integrin is reported to facilitate leukocyte transmigration through the inflammatory endothelium (13). Thus, both receptors are known to play important roles in host defense during an infection. We investigated whether treatment with the PAR2-cAP (1 x 10–4 M) might enhance the known effects of IFN-{gamma} on the cell surface expression of both receptors on human monocytes. Monocytes were treated with the IFN-{gamma} and/or PAR2-cAP during 6 or 12 h. Significant effects were detected only at the 12-h time point. Exposure to PAR2-cAP alone did not lead to a significant change of {alpha}Vβ3 cell surface display as compared with untreated monocytes (Fig. 6A). However, treatment of noninfected monocytes with IFN-{gamma} enhanced {alpha}Vβ3 cell surface expression (MFI increases at ~100% as compared with untreated cells) (Fig. 6A). Further, treatment with a combination of PAR2-cAP and IFN-{gamma} caused an enhancement of cell surface {alpha}Vβ3 compared with cells treated with IFN-{gamma} alone (the MFI increases at 165 ± 19% as compared with control cells) (Fig. 6A). A similar enhancement by PAR2 agonist coapplication was observed for the IFN-{gamma}-induced up-regulation of cell surface expression of CD64 (after application of IFN-{gamma} alone, MFI increased at 54 ± 16% as compared with untreated monocytes; after coapplication of both stimuli, PAR2-cAP and IFN-{gamma}, MFI increased at 94 ± 20% as compared with untreated cells) (Fig. 6B). The treatment of monocytes with PAR2-cRP either alone or in combination with IFN-{gamma} did not have any impact on {alpha}Vβ3 as and CD64 cell surface display (data not shown).


Figure 6
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  		FIGURE 6. Analysis of the changes of {alpha}Vβ3 and CD64 (Fc{gamma}RI) expression on cell surface of human noninfected monocytes after PAR2-cAP, IFN-{gamma}, or combined stimulation was performed by flow cytometry. Monocytes were stimulated for 12 h with 10–4 M cAP, 200 U/ml IFN-{gamma}, or both. Results are presented as average values of stimuli effects at MFI for {alpha}Vβ3 (A) or Fc{gamma}RI (B) as compared with unstimulated monocytes ± SEM. Student’s t test was applied: *, p < 0.05; ##, **, p < 0.01; and ***, p < 0.005. The symbol * marks the significance as compared with control and symbol # as compared with IFN-{gamma} sample. PAR2 agonist is able to enhance IFN-{gamma}-induced up-regulation of {alpha}Vβ3 and Fc{gamma}RI on monocyte cell surface. The number of {alpha}Vβ3 positive cells in unstimulated control samples was 67 ± 8%, and the number of Fc{gamma}RI positive cells was 83 ± 3%. Five independent experiments were performed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The main finding of our work is that combined application of PAR2 agonist along with IFN-{gamma} is able to enhance the anti-viral effects of cytokine on human monocytes: 1) to increase IFN-{gamma}-mediated reduction in viral replication, 2) to increase the release of protective monocyte cytokines like IP-10 that can attract monocytes and T lymphocytes to the site of viral infection, and 3) to increase monocyte surface expression of CD64 (Fc{gamma}RI) and {alpha}Vβ3 that would enhance the opsonization of virus and promote the migration of monocytes across the endothelial barrier to populate the virus-infected tissue. Moreover, the treatment of human monocytes shortly before and during viral infection with synthetic PAR2 agonist alone reduced influenza A replication in these cells. The nature of these results limited the use of trypsin and tryptase, classical PAR2 activating proteases, in our study. Both, trypsin and tryptase could mediate PAR2-independent effects apart from those associated with receptor activation (27, 28). Moreover, trypsin as well as tryptase could directly promote influenza A replication due to the ability of these enzymes to cleave viral hemagglutinin and induce its maturation (20, 21). Thus, taking into account the data of our study, as well as well-documented features of trypsin and tryptase, one might assume that the use of only specific PAR2 agonist, which is not able to affect hemagglutinin maturation, could be beneficial during influenza A infection. Currently, synthetic PAR2 peptide agonist satisfies these conditions. Whether any of the proteases might also meet these conditions should be clarified in future studies. The use of reliable commercial PAR2 antagonist in such studies could also be very helpful, and we hope that such antagonist will be very soon available for the researchers in PAR field.

Human influenza A virus causes a severe disease of the upper respiratory tract. This virus is able to replicate not only in epithelial cells, but also in monocytes/macrophages (29). Replication of human influenza A virus in monocytes promotes viral spreading through a host body. IFNs were used for prophylaxis against experimentally induced influenza A virus infection in humans and mice (30, 31). It was our working hypothesis, based on recently published data (8), that application of PAR2 agonist could also play a role in the suppression of influenza A virus replication. Short pretreatment and further application of stimuli during infection allowed us to mimic such a situation, in which IFNs are applied for prophylaxis against influenza A virus infection. Indeed, under such conditions, both PAR2-cAP and IFN-{gamma} were able to suppress influenza A virus replication in human monocytes. However, only PAR2-cAP pretreatment (in contrast to pretreatment with IFN-{gamma}) was not sufficient for suppression of influenza A virus replication in human monocytes.

The susceptibility of cells to influenza A virus is known to depend on the level of NF-{kappa}B activation (22, 23). Normally, I{kappa}B{alpha} silences NF-{kappa}B activation. The phosphorylation and degradation of I{kappa}B{alpha}, upon cell stimulation, makes NF-{kappa}B free to trigger transcriptional events. The level of cytoplasmic I{kappa}B{alpha}, therefore, may serve as an indirect indicator of the level of active NF-{kappa}B. In our study, the coapplication of PAR2-cAP and IFN-{gamma} enhanced the availability of cytoplasmic I{kappa}B{alpha} at all investigated time points (15 min, 30 min, and 2 h). Therefore, the reduction of influenza A virus replication in human monocytes after coapplication of PAR2-cAP and IFN-{gamma} correlates with enhanced availability of cytoplasmic I{kappa}B{alpha}. This finding suggests that combined PAR2-cAP and IFN-{gamma} stimulation results in a cellular milieu in which the activation of NF-{kappa}B would be attenuated. Such reduction of NF-{kappa}B activation could be one of the reasons for the impact, which coapplication of PAR2 agonist and IFN-{gamma} has on influenza A virus replication in human monocytes.

Huber and colleagues (12) demonstrated a significant contribution of Fc{gamma}RI to anti-viral defense through the ingestion of opsonized influenza A viruses by murine macrophages. Moreover, IFN-{gamma} stimulation enhances the expression of Fc{gamma}RI on the cell surface of human monocytes (32). Thus, we investigated whether PAR2-cAP affects an IFN-{gamma}-induced effect on Fc{gamma}RI display on monocyte cell surface. Indeed, costimulation of noninfected human monocytes with PAR2-cAP and IFN-{gamma} resulted in a significant increase of Fc{gamma}RI surface expression as compared with monocytes treated with either agonist alone. This finding supports our hypothesis that a PAR2 agonist and IFN-{gamma} can work together to enhance anti-viral effects of the cytokine on human monocytes.

We also investigated the ability of PAR2-cAP to act on immunomodulatory functions of IFN-{gamma} in human monocytes. The production of several chemokines increases after IFN-{gamma} treatment (15) or upon influenza A virus infection of human monocytes (9). Among these chemokines are attractants for monocytes and T cells, such as IP-10, MIP-1, MCP-1, and RANTES. IP-10 facilitates endothelial-lymphocyte interactions and transmigration of monocytes as well as T cells toward the site of infection or inflammation (33). Our data demonstrate that PAR2 agonist stimulation results in an increased IP-10 secretion by influenza A-infected human monocytes. Moreover, we showed that the IFN-{gamma}-induced increase of IP-10 release was enhanced by simultaneous coapplication of PAR2-cAP in virus-infected as well as noninfected monocytes. Surprisingly, we also found that the PAR2 agonist amplifies the ability of IFN-{gamma} to increase the cell surface expression of the monocyte adhesion molecule, {alpha}Vβ3. This integrin promotes monocyte transmigration process via interaction with PECAM and vitronectin on endothelial cells (13). Taken together, our findings indicate that coapplication of PAR2 agonist along with IFN-{gamma} is able to enhance not only direct anti-viral, but also immunoregulatory effects of the IFN-{gamma} on functions of human monocytes. In this regard, we also found that IFN-{gamma} stimulation amplifies the cell surface display of PAR2 on human monocytes. These coordinated interactions between IFN-{gamma} and PAR2 agonists therefore represent a mechanism whereby the anti-viral and/or immunoregulatory effects of IFN-{gamma} could be enhanced in human monocytes.

In conclusion, our data clearly demonstrate that PAR2 agonist acting along with IFN-{gamma} could enhance the protective effect of this cytokine on human monocyte function in vitro. Moreover, even in the absence of IFN-{gamma} stimulation, we found that the application of PAR2-cAP shortly before and during viral amplification reduces influenza A virus replication in human monocytes. Together, these major findings of our work indicate that the application of PAR2 agonist might have therapeutic potential in the setting of influenza A virus infection.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Interdisziplinäres Zentrum für Klinische Forschung Münster (Stei2/027/06), German Research Foundation (SFB 293-A14, SFB492-B13, STE 1014/2-2), CERIES (Paris) (to M.S.), SFB 293 (to S.L.), Innovative Medizinische Forschung (University of Münster; Grant SH 120709, to V.M.S.), and Canadian Institutes of Health Research (Operating and Proteinases and Inflammation Network grants to M.D.H.). Back

2 M.F. and V.M.S. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Victoria Shpacovitch, Department of Dermatology and Ludwig-Boltzmann-Institute for Immunobiology of the Skin, University of Münster, von-Esmarch-Strasse 58, 48149 Münster, Germany. E-mail address: shpacovi{at}ukmuenster.de Back

4 Abbreviations used in this paper: PAR2, proteinase-activated receptor 2; IL-1ra, IL-1 receptor antagonist; IP-10, IFN-{gamma}-inducible protein 10 kDa; MFI, mean fluorescence intensity; PAR2-cAP, PAR2-tc-activating peptide; PAR2-cRP, PAR2-tc-reverse peptide; MOI, multiplicity of infection. Back

Received for publication August 9, 2007. Accepted for publication March 13, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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