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The Journal of Immunology, 2007, 179, 6080 -6087
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Monocyte-Specific Peptide from Herpes Simplex Virus Type 2 Glycoprotein G Activates the NADPH-Oxidase but Not Chemotaxis through a G-Protein-Coupled Receptor Distinct from the Members of the Formyl Peptide Receptor Family1

Lars Bellner2,3,*, Jennie Karlsson2,*, Huamei Fu*, François Boulay{dagger}, Claes Dahlgren*, Kristina Eriksson4,* and Anna Karlsson*

* Department of Rheumatology and Inflammation Research, Göteborg University, Göteborg, Sweden; and {dagger} CEA-Grenoble, Grenoble, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have recently identified a peptide derived from the secreted portion of the HSV-2 glycoprotein G, gG-2p20, to be proinflammatory. Based on its ability to activate neutrophils and monocytes via the formyl peptide receptor (FPR) to produce reactive oxygen species (ROS) that down-regulate NK cell function, we suggested it to be of importance in HSV-2 pathogenesis. We now describe the effects of an overlapping peptide, gG-2p19, derived from the same HSV-2 protein. Also, this peptide activated the ROS-generating NADPH-oxidase, however, only in monocytes and not in neutrophils. Surprisingly, gG-2p19 did not induce a chemotactic response in the affected monocytes despite using a pertussis toxin-sensitive, supposedly G-protein-coupled receptor. The specificity for monocytes suggested that FPR and its homologue FPR like-1 (FPRL1) did not function as receptors for gG-2p19, and this was also experimentally confirmed. Surprisingly, the monocyte-specific FPR homologue FPRL2 was not involved either, and the responsible receptor thus remains unknown so far. However, the receptor shares some basic signaling properties with FPRL1 in that the gG-2p19-induced response was inhibited by PBP10, a peptide that has earlier been shown to selectively inhibit FPRL1-triggered responses. We conclude that secretion and subsequent degradation of the HSV-2 glycoprotein G can generate several peptides that activate phagocytes through different receptors, and with different cellular specificities, to generate ROS with immunomodulatory properties.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Herpes simplex virus 2 is a sexually transmitted pathogen that infects the genital tract mucosa and is the most common causative agent of genital ulcer disease in humans. Once HSV-2 infects the epithelium and replicates, it can be transmitted to the sacral ganglia via nerve axons and establish a latent infection, which in association with factors such as stress, fever, UV-irradiation, and immunosuppression can reactivate and cause recurrent disease (1, 2). Professional phagocytes (neutrophil granulocytes and monocytes/macrophages) constitute an important first line of defense against microbial intruders, including many viruses. Their potential role in HSV infection is suggested by studies showing that reduced numbers of granulocytes, or functional defects in these cells, may lead to severe and recurrent infections due to a deficiency in the control of HSV replication (3, 4). Functional restoration of the impaired cells is also associated with a recovery of the infected host (4).

We recently identified an HSV-2 peptide, named gG-2p20, which possesses proinflammatory properties in being an activator of professional phagocytes (5). Based on our results, we introduced a new concept for the role of inflammatory cells in HSV-2 pathogenesis; the fact that gG-2p20-activated phagocytes could impair NK-cell function suggested that this HSV-2-derived proinflammatory peptide may contribute to the survival of virus-infected cells and thereby be of immunomodulatory importance in HSV-2 pathogenesis (5). We were also able to identify both the receptor through which the HSV-2 peptide triggered its proinflammatory activity and the mechanism by which the NK cell function was impaired. The triggering receptor was identified as one of the classical pertussis toxin sensitive G-protein coupled chemoattractant receptors (GPCR)5 known as the formyl peptide receptor (FPR) (6). This is a high-affinity pattern recognition receptor with the ability to track not only formylated peptides released from bacteria, but also virus-derived proteins, e.g., peptides from the envelope proteins of HIV-1 (7) or the earlier mentioned peptide from HSV-2 (5), as well as a number of different endogenous agonists (8). In addition to FPR, there are two other members of the formyl peptide receptor family, formyl peptide receptor like-1 and -2 (FPRL1, FPRL2). The FPR family members are differently expressed in phagocytes, with neutrophils expressing FPR and FPRL1, and monocytes/macrophages expressing all three. A number of agonists that trigger cells through FPRL1 have been described whereas the monocyte-specific family member FPRL2 was until very recently considered an orphan receptor (9).

Ligation of the FPR family receptors with specific agonists (including gG-2p20 for FPR) results in oxygen radical generation by activation of the NADPH-oxidase, an electron transport system assembled in the plasma membrane during cell activation in both neutrophils and monocytes/macrophages (10). The primary products of the assembled oxidase are superoxide anions and hydrogen peroxide, and these reactive oxygen species (ROS) can function as regulators of other immune reactive cells, providing a molecular explanation behind the functional changes seen as apoptotic/necrotic processes in NK cells exposed to activated phagocytes (11, 12, 13, 14).

The HSV-2 glycoprotein G, from which the proinflammatory peptide gG-2p20 was derived, is the only HSV-2 envelope protein to be cleaved post-translationally, generating two proteins of which one has the unique property of being secreted from the infected cells (sgG-2) (15, 16, 17, 18). Previous to our study of the sgG-2-derived peptide, indicating proinflammatory activity, the secreted protein had not been ascribed a specific function. In this study, we have continued our investigation on the immunomodulatory properties of peptides derived from the sgG-2 amino acid sequence. In addition to the earlier identified neutrophil/monocyte activating peptide, we found one peptide, gG-2p19, which induced NADPH-oxidase activation in monocytes, but not neutrophils. Although the activity induced by gG-2p19 was monocyte specific and mediated through a pertussis toxin-sensitive GPCR, it was not triggered by FPRL2 (nor by FPR or FPRL1), which is the only member of the FPR family that is expressed solely in monocytes. Intriguingly, gG-2p19 was not a monocyte chemoattractant, suggesting that the receptor involved has a more restricted functional spectrum compared with other GPCRs.


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

Three 15-mer synthetic peptides (Fig. 1A) from the secreted portion of the HSV-2 glycoprotein G (sgG-2) of strain HG52 (19) were synthesized as described earlier using F-moc synthesis chemistry (20) and were from two different sources, KJ Ross-Petersen (used in Fig. 1) and GenScript (used in Figs. 2–8). The purity of the peptides was found to be ≥95% by reverse phase HPLC. The HSV-2 gG-2 peptides were dissolved in either water, sodium bicarbonate (NaHCO3 0.02 M in H2O), or DMSO to 6 mM and stored at –70°C until use.


Figure 1
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  		FIGURE 1. The gG-2p19 induces activation of the monocyte (but not neutrophil) NADPH-oxidase. A, Schematic representation of the amino acid sequences of gG-2p19, gG-2p20, and gG-2p21. B and C, Monocytes (solid lines) and neutrophils (dashed lines) were activated with gG-2p20 (30 µM) (B) and gG-2p19 (30 µM) (C) and the following superoxide anion release was measured continuously for 4 min by isoluminol-ECL (CL). The amount of superoxide anion is presented as 106 cpm (Mcpm). Arrows indicate the time of addition of peptide. The figure shows one representative experiment of three. The amount of superoxide released from monocytes in response to different concentrations of gG-2p19 was measured (n = 3–4; mean ± SD) (C, inset). No NADPH-oxidase activity was detected for gG-2p21 (data not shown).

 

Figure 2
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  		FIGURE 2. The gG-2p19-induced NADPH-oxidase activation in monocytes is pertussis toxin sensitive. Monocytes were preincubated at 37°C for 90 min in the absence (solid line), or presence (dashed line) of pertussis toxin (PT, 500 ng/ml) and were then stimulated with gG-2p19 (30 µM). The superoxide anion release was measured continuously for 6 min, by isoluminol-enhanced CL. To ensure that the PT treatment was successful in inhibiting GPCR-triggered NADPH-oxidase activation without affecting the overall condition of the cells, control experiments were performed (inset). The PT treatment successfully inhibited the fMLF-induced response (0.1 µM), while the PT-insensitive PMA-induced response (50 nM) was unaffected. The amount of superoxide anion is presented as 106 cpm (Mcpm). The figure shows one representative experiment of three.

 

Figure 3
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  		FIGURE 3. The gG-2p19 does not induce chemotaxis in monocytes. Monocyte transmigration in response to gG-2p19 (•), gG-2p20 ({circ}), buffer ({square}), and fMLF (10 nM, {blacksquare}) was assayed using a ChemoTX multiwell chamber system. Migration was determined after 90 min incubation by measuring the amount of myeloperoxidase in lysates of migrated cells. Data are expressed as mean per cent migrated cells ± SEM for different peptide concentrations, where maximum is the OD450 value obtained from lysates of 105 monocytes added directly to the lower chamber.

 

Figure 4
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  		FIGURE 4. Desensitization of the gG-2p19-induced oxidative response. Monocytes were preactivated with gG-2p19 (30 µM; A), fMLF (0.1 µM; B), or WKYMVM (0.1 µM; C), and subsequently challenged with gG-2p19 (30 µM). The oxidative response was followed by isoluminol-amplified CL. The curves show that gG-2p19 is homologously desensitized (A) but not heterologously desensitized by neither fMLF (agonist for FPR; B) or WKYMVM (agonist for FPRL1/FPRL2; C). The amount of superoxide anion is presented as 106 cpm (Mcpm). The figure shows one representative experiment of three.

 

Figure 5
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  		FIGURE 5. Effect of receptor antagionists and inhibitors on the monocyte NADPH-oxidase response to gG-2p19. Cells were preincubated at 37°C for 5 min in the absence (solid line) or presence (dashed line) of the FPR antagonist N-t-Boc-MLF (10 µM; dashed line), the FPR inhibitor Cyclosporin H (0.25 µM; dotted line), or the FPRL1 antagonist WRW4 (0,5 µM; thin line) and were subsequently stimulated with fMLF (4 nM; A), WKYMVM (4 nM; B), or gG-2p19 (30 µM; C). The amount of superoxide anion is presented as 106 cpm (Mcpm). The figure shows one representative experiment of three.

 

Figure 6
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  		FIGURE 6. The gG-2p19 is not a ligand for any of the formyl peptide receptor family receptors. Fura-2 loaded nondifferentiated HL-60 cells (106/ml) overexpressing either FPR (A), FPRL1 (B), FPRL2 (C), or monocytes (D; 106/ml) were analyzed for mobilization of intracellular Ca2+ in response to gG-2p19 (30 µM; bold lines). The FPR/FPRL1/FPRL2 agonists fMLF and WKYMVM (0.1 µM) were used as positive controls (thin lines). Representative Ca2+ measurements (n = 3) are shown, given as the relative change in Ex340/Ex380 ratio.

 

Figure 7
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  		FIGURE 7. The monocyte NADPH-oxidase response to gG-2p19 is inhibited by the gelsolin-derived peptide PBP10. Monocytes were preincubated at 37°C for 5 min in the absence (solid line), or presence (dashed line) of PBP10 (1 µM; inhibitor of the FPRL1-, but not the FPR-induced signaling pathway) and were subsequently stimulated with WKYMVM (0.1 µM; A), fMLF (0.1 µM; B), or gG-2p19 (30 µM; C).The amount of superoxide anion is presented as 106 cpm (Mcpm). The figure shows one representative experiment of three.

 

Figure 8
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  		FIGURE 8. Desensitization of the fMLF and WKYMVM responses by gG-2p19. Monocytes were preactivated with gG-2p19 (30 µM; added on ice and incubated at 37°C for 10 min) and subsequently challenged with fMLF (0.1 µM; A) or WKYMVM (0.1 µM; B). The oxidative response was followed by isoluminol-amplified CL. The curves show that gG-2p19 partially desensitized the fMLF-induced response, whereas the WKYMVM-induced response was unaltered. The amount of superoxide anion is presented as 106 cpm (Mcpm). The figure shows one representative experiment of three.

 
The hexapeptides WKYMVM and WKYMVm, which were synthesized and HPLC purified by KJ Ross-Petersen, and the formylated tripeptide formyl-methionyl-leucyl-phenylalanine (fMLF), which was purchased from Sigma-Aldrich, were dissolved in DMSO (0.01 M in H2O) to 0.1 mM and stored at –70°C until use. Subsequent dilutions of all peptides were made in Krebs-Ringer phosphate buffer (KRG (pH 7.3); 120 mM NaCl, 5 mM KCl, 1.7 mM KH2PO4, 8.3 mM NaH2PO4, and 10 mM glucose) supplemented with Ca2+ (1 mM) and Mg2+ (1.5 mM).

Cyclosporin H was provided by Novartis Pharma. Ficoll-Paque was obtained from Amersham Biosciences N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine (N-t-Boc-MLF), isoluminol, and PMA were obtained from Sigma-Aldrich, and HRP was obtained from Boehringer Mannheim. The WRW4 was from GenScript. The gelsolin-derived peptide PBP10 (residues 160–169, aa sequence QRLFQVKGRR) was prepared by solid phase peptide synthesis and coupled to rhodamine as described earlier (21).

Separation of human monocytes

Human PBMC were separated from buffy coats from healthy blood donors. After Ficoll-Paque centrifugation, mononuclear cells were separated into lymphocytes and monocytes using counter current centrifugal elutriation as described in detail elsewhere (22). This procedure yielded a fraction with >90% monocytes at a flow rate of 20–22 ml/min. The cells were washed and resuspended in KRG and stored on melting ice until use.

Separation of human neutrophils

Human peripheral blood neutrophils were isolated from buffy coats from healthy blood donors using dextran sedimentation and Ficoll-Paque gradient centrifugation as described (23). The cells were washed and resuspended in KRG and stored on melting ice until use.

Stable expression of FPR, FPRL1, and FPRL2 in undifferentiated HL-60 cells

The stable expression of FPR, FPRL1, and FPRL2 in undifferentiated HL-60 cells has been described previously (24, 25). Transfected cells were cultured in RPMI 1640 (PAA Laboratories GmbH) containing FCS (10%) (PAA Laboratories GmbH), penicillin/streptomycin (1%) (PAA Laboratories GmbH), and G418 (1 mg/ml). The maximal density was maintained below 2 x 106 cells/ml. The cells were passaged to a concentration of 5 x 105 cells/ml ~24 h before use in assays.

Measurement of superoxide release

The NADPH-oxidase activity was determined using isoluminol-ECL chemiluminescence (CL) (26). The CL activity was measured in a six-channel Biolumat LB 9505 (Berthold), using disposable 4-ml polypropylene tubes with a 400-µl reaction mixture containing 4 x 104 cells. The tubes were equilibrated in the Biolumat for 5 min at 37°C, after which the stimulus (20 µl) was added. The light emission was recorded continuously, and the light intensity was measured 15 times per minute. The relation between the amount of superoxide produced and the amount of detected light is dependent on the luminometer, but it can easily be determined by a direct comparison of the superoxide dismutase-inhibitable reduction of cytochrome c with the integrated value of the superoxide dismutase-inhibitable chemiluminescence (27, 28). With the equipment used, 7.2 x 107 counts were found to correspond to the production of 1 nmol superoxide (using a millimolar extinction coefficient for cytochrome c of 21.1).

When experiments were performed with antagonists and inhibitors such as N-t-Boc-MLF, cyclosporin H, WRW4, or PBP10, these substances were included in the reaction mixture during the 5 min equilibration period. In desensitization experiments, the primary stimulus was added to the reagent tubes on ice or at 37°C, followed by incubation in the Biolumat for 10 min before addition of the second stimulus.

Monocyte chemotaxis

Monocyte chemotaxis was determined using ChemoTX multiwell chambers (Neuro Probe) with a membrane pore diameter of 5 µm, according to instructions provided by the manufacturer. Cells were allowed to migrate through the membranes toward stimulus added to the lower chambers during a 90-min incubation at 37°C, after which the cells in the lower chamber were lysed with 0.2% cetyltrimethylammonium bromide in PBS with 0.2% BSA. Twenty microliters of each lysate were transferred into a 96-well ELISA plate (MaxiSorp, Nunc) and quantified based on myeloperoxidase activity by the addition of 45 µl peroxidase substrate solution containing 0.1 mg/ml tetramethylbenzidine and 0.04% H2O2 in 0.2 M phosphate-citrate buffer (pH 5). The reaction was stopped after 2 h by the addition of 25 µl 1M H2SO4 and the absorbance (OD) was measured at 450 nm. Relative migration (%) was obtained by dividing the sample OD450 with the OD450 of cells added directly to the lower compartment of the multiwell chamber (representing 100% migration). Spontaneous movement, i.e., OD450 values obtained from samples with no added stimulus, was used as negative control. As positive control, migration toward fMLF (10–8 M) and gG-2p20 (varying concentrations) were used. The OD450 values used are means of duplicates.

Cytosolic calcium mobilization

Monocytes or undifferentiated HL-60 cells (2 x 107/ml) stably expressing FPR, FPRL1 (26), or FPRL2 (29) were incubated with fura-2AM (2 µM) in calcium free KRG supplemented with BSA (0.1%) at room temperature for 30 min in the dark. Cells were then diluted to twice the original volume with RPMI 1640 culture medium without phenol red (PAA Laboratories GmbH) and centrifuged. The cells were washed once and resuspended to a density of 2 x 107/ml in KRG containing 1.0 mM Ca2+. Cells were equilibrated for 5 min at 37°C, after which gG-2p19 or positive control peptides (fMLF for FPR and WKYMVm for FPRL1 and FPRL2) were added. The fura-2 fluorescence was measured by a luminescence spectrometer (LS50B; PerkinElmer) using excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. Maximal and minimal fluorescence were determined in the presence of Triton X-100 and EGTA/Tris-HCl, respectively. The intracellular calcium levels are shown as Ex340:Ex380 ratios (25).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
An HSV-2 glycoprotein G-derived peptide triggers the monocyte NADPH-oxidase to produce superoxide anions

Through screening of peptides spanning the whole amino acid sequence of the secreted portion of HSV-2 glycoprotein G, we have previously identified a peptide, gG-2p20, that exerted downregulatory functions on human NK cells by activating phagocytes to produce ROS (5). In this study, we analyze two neighboring peptides derived from the same protein, overlapping the gG-2p20 sequence with 5 aa in either end of the peptide, and extended with 10 aa to the N-terminal side (gG-2p19) or the C-terminal side (gG-2p21) (Fig. 1A). When exposing neutrophils or monocytes to these peptides we found that whereas gG-2p20 activated a response in both monocytes and neutrophils (Fig. 1B), gG-2p19 activated a respiratory burst response in monocytes but was without effect on neutrophils (Fig. 1C). The gG-2p19-induced oxidase activation in monocytes was concentration dependent (Fig. 1C, inset). No respiratory burst activity was triggered by gG-2p21 in either of the cells (data not shown).

The NADPH-oxidase activity triggered in monocytes by gG-2p19 is pertussis toxin sensitive

The gG-2p19 induced production of ROS in monocytes (Fig. 1 and 2) with a time course resembling that induced by the earlier described FPR agonists gG-2p20 (Fig. 1 and (5)) and fMLF (Fig. 2; inset). The ROS production induced by gG-2p19 was inhibited by preincubation of the monocytes with pertussis toxin, known to specifically block signaling induced by Gi GPCRs (30), suggesting that also gG-2p19 induces activation through a GPCR.

The gG-2p19 is not chemotactic for monocytes

The signals generated by GPCRs in phagocytes usually mediate a chemotactic response. No such response was however triggered by the gG-2p19 peptide, irrespective of the concentration used to trigger the response (Fig. 3). The earlier-described FPR ligands fMLF and gG-2p20 were used as controls, both inducing a potent chemotactic response in monocytes (Fig. 3).

The gG-2p19 does not interact with FPR or FPRL1

The gG-2p19-induced NADPH-oxidase response closely resembled responses induced by other peptides (bacterial and synthetic) acting through different GPCRs, including those of the FPR family. This, together with the fact that the earlier-described HSV-2 peptide gG-2p20 is an FPR agonist, gave us cause to investigate the involvement of FPR family members in the gG-2p19-induced monocyte response. The basis for the first part of this investigation was desensitization protocols using gG-2p19, the FPR agonist fMLF, and the FPRL1/FPRL2 agonist WKYMVM. In these experiments we exploit the fact that agonist binding to a particular receptor induces receptor-specific desensitization and subsequent inability of the cells to respond to the same or a second agonist that ligates the same receptor (29).

Attempts to challenge gG-2p19-pretreated monocytes with the same agonist a second time failed, meaning that the HSV-2 peptide acts through a receptor that can be homologously desensitized (Fig. 4A). Cells pretreated with the FPR agonist fMLF were however still able to mount an oxidative response when stimulated with gG-2p19 (Fig. 4B), i.e., fMLF did not heterologously desensitize the cells to the HSV-2 peptide. Furthermore, preincubation of the cells with the FPR receptor antagonists N-t-Boc-MLF or cyclosporin H, both of which completely abrogate fMLF-induced responses (Fig. 5A), did not prevent the cells from responding to gG-2p19 (Fig. 5C), even though both N-t-Boc-MLF and cyclosporin H pretreatment did affect the magnitude of the gG-2p19-induced responses (Fig. 5C). Taken together, we conclude that FPR is not involved in the response to gG-2p19.

No substantial desensitization of the gG-2p19-induced activation could be seen in monocytes first treated with the FPRL1/FPRL2 agonist WKYMVM (Fig. 4C), suggesting that the peptide uses neither FPRL1 nor FPRL2. For FPRL1, this is supported by inhibition experiments using the FPRL1-specific antagonist WRW4 (Fig. 5B), which did not prevent a gG-2p19-induced response (Fig. 5C).

The gG-2p19 does not trigger any response in transfected cells expressing FPRL2

Because gG-2p19 was a potent activator of the NADPH-oxidase in monocytes (expressing FPR, FPRL1, and FPRL2) but not in neutrophils (which express FPR and FPRL1), FPRL2 was more plausible as a receptor for gG-2p19 than FPR and FPRL1. To determine whether gG-2p19 could activate cells specifically through FPRL2, we investigated the triggering potential of the peptide in HL-60 cells stably transfected to express one or the other of the three FPR family members. In accordance with our desensitization data, gG-2p19 could not trigger any transient Ca2+ response in cells expressing either FPR (Fig. 6A) or FPRL1 (Fig. 6B). The same was true also for FPRL2, i.e., gG-2p19 could not activate the FPRL2-expressing cells (Fig. 6C). Undifferentiated HL-60 cells transfected with an irrelevant receptor, CXCR2, do not show a Ca2+ response to FMLF or WKYMVm (25). In contrast, gG-2p19 did induce a Ca2+ response in purified monocytes (Fig. 6D). All taken together, our data indicate that gG-2p19 interacts with a GPCR that is not a member of the formyl peptide receptor family.

The gG-2p19 activation of monocytes is inhibited by PBP10

We have recently shown that the signaling cascades induced in neutrophils by agonists binding to FPR and FPRL1 differ in their sensitivity to the gelsolin-derived, PIP2-binding peptide PBP10 (31). This membrane-permeable gelsolin-derived peptide specifically inhibits signaling through FPRL1. In an attempt to determine whether the signaling route induced by the gG-2p19 receptor was in any way similar to that induced by FPRL1, we investigated the inhibitory effect of PBP10 on the gG-2p19-induced response in monocytes. The inhibitory profile of PBP10 with respect to FPR and FPRL1 was the same in monocytes as in neutrophils, i.e., the response induced in monocytes by WKYMVM (FPRL1/FPRL2 agonist) was completely inhibited in presence of PBP10 (Fig. 7A), whereas the response induced by fMLF (FPR agonist) was unaffected (Fig. 7B). Interestingly, the monocyte NADPH-oxidase response induced by gG-2p19 was substantially inhibited in the presence of PBP10 (Fig. 7C), indicating that the gG-2p19-binding GPCR shares the PBP10 sensitive signaling route with FPRL1.

Receptor cross-talk and hierarchy

There is a hierarchical receptor cross-talk within the phagocyte GPCR family. Cells desensitized to FPR or FPRL1 agonists are down-regulated not only in the response to their respective receptor-specific agonists but also to a second stimulation with the GPCR agonists IL-8 or platelet-activating factor (32, 33). The fact that desensitization of FPR or FPRL1 does not affect the gG-2p19-induced response suggests not only that monocyte activation by gG-2p19 does not involve the FPR family receptors, but also that the gG-2p19 receptor is not subordinated FPR or FPRL1. However, desensitization experiments, in which the order of the agonists was changed, revealed receptor cross-talk between the gG-2p19 receptor and FPR. We found that the FPR-triggered response was reduced when monocytes were desensitized with gG-2p19 before stimulation with fMLF (Fig. 8A). No such cross-talk or inhibition was seen when fMLF was replaced by the FPRL1 agonist WKYMVM (Fig. 8B). Our interpretation of these results is that activation of the gG-2p19 receptor cross-desensitizes the FPR, i.e., that the gG-2p19 receptor is hierarchically above the FPR. The fact that FPRL1 differs from FPR by being unaffected by desensitization of the gG-2p19 receptor gives support to the notion that these two receptors, although closely related, differ in their intracellular signaling.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A peptide derived from the secreted portion of HSV-2 glycoprotein G has been identified that selectively activates monocytes through a pertussis toxin-sensitive receptor. The peptide, denoted gG-2p19, triggered activation of the superoxide-generating NADPH-oxidase, but was not able to induce monocyte chemotaxis. The main receptor candidate, FPRL2, the monocyte-specific member of the formyl peptide receptor family, was found not to be involved in cell activation. The peptide used a receptor distinct from the formyl peptide receptor family, but the receptor appeared to share at least one crucial step in its signaling pathway with one of the family members, FPRL1, based on the inhibition of the response to gG-2p19 by the PIP2-binding, gelsolin-derived peptide PBP10, known to block FPRL1 signaling (31).

The strategy used to identify gG-2p19 as a monocyte-activating peptide has been used earlier in the search for neutrophil-activating peptides from the HSV-2 glycoprotein G (5). We then identified gG-2p20 as a chemotactic peptide for both neutrophils and monocytes and we were also able to disclose the identity of the gG-2p20 binding receptor, which was the FPR. We originally used thirty-three 15-mer, partly overlapping peptides covering the entire secreted portion of the HSV-2 glycoprotein G. The fact that the only two proinflammatory peptides, gG-2p19 and gG-2p20, originate from the same part of the protein and contain an identical sequence of five amino acids, suggested to us that they should bind to two closely related receptors.

Because gG-2p19 did not activate neutrophils, the receptor could be differently expressed in neutrophils and monocytes, which made FPRL2 the most obvious candidate. The FPRL2 was originally cloned from a promyelocyte cDNA library using low-stringency hybridization with the FPR cDNA as a probe (34, 35), and it was for a long time regarded as an orphan receptor. In contrast to the other two members of the receptor family (FPR and FPRL1), the expression of FPRL2 is restricted to cells in the monocyte/macrophage linage.

From our data we conclude, however, that the activating signal in monocytes induced by gG-2p19 is not transduced through FPRL2. This conclusion is based on the facts that no signal is generated by gG-2p19 in HL-60 cells transfected to express FPRL2 and that the FPRL2 agonist WKYMWM did not desensitize monocytes to the gG-2p19 peptide. The fact that no activity is induced in FPRL2-expressing HL-60 cells by gG-2p19 strongly indicates that another monocyte-specific receptor is involved, but desensitization or binding inhibition experiments with an FPRL2-specific ligand would of course add strength to the conclusion. Such an agonist has been described (9). We have tried to use the FL2 peptide (with the sequence MLGMIKNSLFGSVETWPWQVL), however, in our experimental systems with FPRL2-expressing cells or monocytes this peptide was without effect (our unpublished data). We can at present only speculate on the reason for this but it has been shown that FL2 induces a strong response in around 50% of the blood cell donors suggesting that we might have obtained all our samples from nonresponders. More importantly, however, is that these cells still responded to gG-p2–19. We cannot at present explain why FL2 fail to trigger a response in FPRL2 expressed in HL-60 cells, but the receptor might look a bit different when expressed in another cell line such as cho-K1 cells. Even though the identity of the gG-2p19 receptor remains to be disclosed, our results suggest that it is a receptor distinct from FPRL2.

However, we have clues to some characteristics of the gG-2p19 receptor. Firstly, the receptor is a pertussis toxin-sensitive receptor. Pertussis toxin inhibits the Gi group of GPCRs in phagocytes, and cell activation through members of this receptor family normally provokes various biochemical or functional responses which contribute to the defense as well as the immunoregulatory functions of these cells. It is well known that the signals underlying the chemotactic behavior differ from those that regulate granule mobilization and secretory events (36). This is clearly illustrated by the fact that the LTB4-specific GPCR has the capacity to transduce ligand binding into a chemotactic response without any concomitant secretion of ROS (37). Furthermore, depending on the structure of the ligand, the occupied FPR can transfer the chemotactic signal alone, or combined with the signal responsible for superoxide secretion (36). We found that the gG-2p19 peptide was able to induce ROS production without triggering a chemotactic response, a combination of signals unique for this receptor-ligand pair. Whether the receptor entirely lacks the ability to mediate a chemotactic response, or if the ligand (gG-2p19) triggers a very specific signaling pathway that bypasses the route of chemotaxis, cannot be determined until other ligands for the receptor has been identified.

Secondly, the unknown receptor shares signaling properties with FPRL1 in that it is sensitive to the inhibitory activity of PBP10, a membrane-permeable polyphosphoinositide-binding peptide derived from the cytoskeletal protein gelsolin (21). We have previously shown that PBP10 blocks FPRL1-induced ROS secretion in neutrophils, while having no effect on the neutrophil response to agonists for FPR, C5aR, PAFR, or CXCR (31). The precise mechanism by which PBP10 selectively interferes with signaling by some receptors but not by others remains to be determined in detail, but it is reasonable to believe that the signaling intracellular parts of the two sensitive receptors, FPRL1 and the gG-2p19 receptor, contain structural similarities.

Thirdly, the gG-2p19 receptor cross-talks with other phagocyte GPCRs. The process known as desensitization (homologous as well as heterologous) is most probably important for the limitation or termination of the cellular response to high concentrations of a chemoattractant, avoiding prolonged activation and by that limiting the inflammation-associated tissue damage at an inflammatory site (8). With respect to cross-talk hierarchy, it is obvious that some activators are strong whereas others are weak. The fact that ligation of the gG-2p19 receptor desensitized the hierarchically strong FPR, indicates that the novel receptor is even stronger. Further desensitization studies have to be performed to more precisely place this receptor in the GPCR hierarchy. It should also be kept in mind that yet other gG-2p19 receptor(s) may be present that is responsible for inhibitory signal(s). We have shown earlier this to be the case for a peptide derived from the N terminus of annexin I, a peptide that triggers NADPH-oxidase activation through one receptor, concomitantly with eliciting inhibitory signals through another receptor (38).

We can only speculate on whether gG-2p19 could exert any effects in vivo. However, there should be high levels of sgG-2 at sites with massive local viral replication, i.e., in herpetic lesions, where >1010 viral particles are found to be present (5). In such an environment, gG-2p19-induced ROS has the potential to eliminate viral particles, but could also lead to local tissue destruction, enhancing the damage induced by the virus itself. However, ROS secreted from neutrophils and monocytes also have a strong negative impact on cell-mediated immunity. Numerous studies have shown that both the cytotoxic capacity and the survival of NK cells and CTL are impaired in the presence of activated phagocytes, in a process that is reversed by ROS scavengers (11, 12, 13, 14). Thus, sgG-2 released from HSV-2-infected cells could, through its interaction with a GPCR (as suggested in this study and in (5)) and subsequent activation of the NADPH-oxidase, contribute to the documented down-regulation of the cellular immune response seen during HSV infection (39, 40, 41).

In summary, we have identified a peptide derived from HSV-2 glycoprotein G that induces release of ROS from monocytes through binding to a GPCR. In vitro experiments showed that the gG-2p19 peptide did not induce a chemotactic response, indicating that the receptor involved has a more restricted functional spectrum compared with other GPCRs. Furthermore, we were able to show that the gG-2p19-interacting GPCR shares at least one common signaling pathway with FPRL1 because both these receptors, as opposed to, for instance, FPR, are sensitive to the PIP2-specific gelsolin-derived peptide PBP10. This is of considerable interest for further elucidation of chemoattractant signal transduction, because FPRL1 is a chemotactic receptor whereas the gG-2p19-binding GPCR is not.


   Acknowledgment
 
We thank Marie-Louise Landelius for excellent technical assistance.


   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 the Swedish Medical Research Council (including a Senior Researcher position for K.E.), the King Gustaf V 80-Year Foundation, the Torsten and Ragnar Söderberg’s foundation, the Göteborg Rheumatism Association, and the Swedish state under the ALF-agreement. Back

2 L.B. and J.K. have contributed equally to the present work. Back

3 Current address: Department of Pharmacology, New York Medical College, Valhalla, NY 10595 Back

4 Address correspondence and reprint requests to Dr. Kristina Eriksson, Department of Rheumatology and Inflammation Research, Guldhedsgatan 10A, 413 46 Göteborg, Sweden. E-mail address: kristina.eriksson{at}microbio.gu.se Back

5 Abbreviations used in this paper: GPCR, G-protein coupled chemoattractant receptors; CL, chemiluminiscence; FPR, formyl peptide receptor; FPRL1, FPR like-1; FPRL2, FPR like-2; KRG, Krebs-Ringer phosphate buffer; N-t-Boc-MLF, N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine; ROS, reactive oxygen species; sgG-2, secreted portion of HSV-2 glycoprotein G. Back

Received for publication August 18, 2006. Accepted for publication August 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Wald, A., J. Zeh, S. Selke, R. L. Ashley, L. Corey. 1995. Virologic characteristics of subclinical and symptomatic genital herpes infections. N. Engl. J. Med. 333: 770-775. [Abstract/Free Full Text]
  2. Wald, A., J. Zeh, S. Selke, T. Warren, A. J. Ryncarz, R. Ashley, J. N. Krieger, L. Corey. 2000. Reactivation of genital herpes simplex virus type 2 infection in asymptomatic seropositive persons. N. Engl. J. Med. 342: 844-850. [Abstract/Free Full Text]
  3. Milligan, G. N.. 1999. Neutrophils aid in protection of the vaginal mucosae of immune mice against challenge with herpes simplex virus type 2. J. Virol. 73: 6380-6386. [Abstract/Free Full Text]
  4. Altamura, M., M. G. Geronimo, L. Nappi, O. Ceci, P. Loizzi, E. Jirillo. 1997. Successful treatment of herpes simplex virus (HSV) recurrent genital infection with recombinant human (rh) granulocyte-macrophage colony stimulating factor (GM-CSF): a case report. Immunopharmacol. Immunotoxicol. 19: 425-436. [Medline]
  5. Bellner, L., F. Thoren, E. Nygren, J. A. Liljeqvist, A. Karlsson, K. Eriksson. 2005. A proinflammatory peptide from herpes simplex virus type 2 glycoprotein G affects neutrophil, monocyte, and NK cell functions. J. Immunol. 174: 2235-2241. [Abstract/Free Full Text]
  6. Le, Y., P. M. Murphy, J. M. Wang. 2002. Formyl-peptide receptors revisited. Trends Immunol. 23: 541-548. [Medline]
  7. Su, S. B., W. H. Gong, J. L. Gao, W. P. Shen, M. C. Grimm, X. Deng, P. M. Murphy, J. J. Oppenheim, J. M. Wang. 1999. T20/DP178, an ectodomain peptide of human immunodeficiency virus type 1 gp41, is an activator of human phagocyte N-formyl peptide receptor. Blood 93: 3885-3892. [Abstract/Free Full Text]
  8. Fu, H., J. Karlsson, J. Bylund, C. Movitz, A. Karlsson, C. Dahlgren. 2006. Ligand recognition and activation of formyl peptide receptors in neutrophils. J. Leukocyte Biol. 79: 247-256. [Free Full Text]
  9. Migeotte, I., E. Riboldi, J. D. Franssen, F. Gregoire, C. Loison, V. Wittamer, M. Detheux, P. Robberecht, S. Costagliola, G. Vassart, et al 2005. Identification and characterization of an endogenous chemotactic ligand specific for FPRL2. J. Exp. Med. 201: 83-93. [Abstract/Free Full Text]
  10. Sheppard, F. R., M. R. Kelher, E. E. Moore, N. J. McLaughlin, A. Banerjee, C. C. Silliman. 2005. Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation. J. Leukocyte Biol. 78: 1025-1042. [Abstract/Free Full Text]
  11. Hellstrand, K., S. Hermodsson. 1993. Serotonergic 5-HT1A receptors regulate a cell contact-mediated interaction between natural killer cells and monocytes. Scand. J. Immunol. 37: 7-18. [Medline]
  12. Djordjevic, V. B.. 2004. Free radicals in cell biology. Int. Rev. Cytol. 237: 57-89. [Medline]
  13. Betten, A., C. Dahlgren, S. Hermodsson, K. Hellstrand. 2003. Histamine inhibits neutrophil NADPH oxidase activity triggered by the lipoxin A4 receptor-specific peptide agonist Trp-Lys-Tyr-Met-Val-Met. Scand. J. Immunol. 58: 321-326. [Medline]
  14. Betten, A., C. Dahlgren, U. H. Mellqvist, S. Hermodsson, K. Hellstrand. 2004. Oxygen radical-induced natural killer cell dysfunction: role of myeloperoxidase and regulation by serotonin. J. Leukocyte Biol. 75: 1111-1115. [Abstract/Free Full Text]
  15. Dall’Olio, F., N. Malagolini, G. Campadelli-Fiume, F. Serafini-Cessi. 1987. Glycosylation pattern of herpes simplex virus type 2 glycoprotein G from precursor species to the mature form. Arch. Virol. 97: 237-249. [Medline]
  16. Balachandran, N., L. M. Hutt-Fletcher. 1985. Synthesis and processing of glycoprotein gG of herpes simplex virus type 2. J. Virol. 54: 825-832. [Abstract/Free Full Text]
  17. Marsden, H. S., A. Buckmaster, J. W. Palfreyman, R. G. Hope, A. C. Minson. 1984. Characterization of the 92,000-dalton glycoprotein induced by herpes simplex virus type 2. J. Virol. 50: 547-554. [Abstract/Free Full Text]
  18. Su, H. K., R. Eberle, R. J. Courtney. 1987. Processing of the herpes simplex virus type 2 glycoprotein gG-2 results in secretion of a 34,000-Mr cleavage product. J. Virol. 61: 1735-1737. [Abstract/Free Full Text]
  19. McGeoch, D. J.. 1987. The genome of herpes simplex virus: structure, replication and evolution. J. Cell Sci. Suppl. 7: 67-94. [Medline]
  20. Levi, M., U. Ruden, B. Wahren. 1996. Peptide sequences of glycoprotein G-2 discriminate between herpes simplex virus type 2 (HSV-2) and HSV-1 antibodies. Clin. Diagn. Lab. Immunol. 3: 265-269. [Medline]
  21. Cunningham, C. C., R. Vegners, R. Bucki, M. Funaki, N. Korde, J. H. Hartwig, T. P. Stossel, P. A. Janmey. 2001. Cell permeant polyphosphoinositide-binding peptides that block cell motility and actin assembly. J. Biol. Chem. 276: 43390-43399. [Abstract/Free Full Text]
  22. Hansson, M., A. Asea, U. Ersson, S. Hermodsson, K. Hellstrand. 1996. Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites. J. Immunol. 156: 42-47. [Abstract]
  23. Boyum, A., D. Lovhaug, L. Tresland, E. M. Nordlie. 1991. Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality. Scand. J. Immunol. 34: 697-712. [Medline]
  24. Christophe, T., A. Karlsson, M. J. Rabiet, F. Boulay, C. Dahlgren. 2002. Phagocyte activation by Trp-Lys-Tyr-Met-Val-Met, acting through FPRL1/LXA4R, is not affected by lipoxin A4. Scand. J. Immunol. 56: 470-476. [Medline]
  25. Dahlgren, C., T. Christophe, F. Boulay, P. N. Madianos, M. J. Rabiet, A. Karlsson. 2000. The synthetic chemoattractant Trp-Lys-Tyr-Met-Val-DMet activates neutrophils preferentially through the lipoxin A(4) receptor. Blood 95: 1810-1818. [Abstract/Free Full Text]
  26. Dahlgren, C., A. Karlsson. 1999. Respiratory burst in human neutrophils. J. Immunol. Methods 232: 3-14. [Medline]
  27. Foyouzi-Youssefi, R., F. Petersson, D. P. Lew, K. H. Krause, O. Nusse. 1997. Chemoattractant-induced respiratory burst: increases in cytosolic Ca2+ concentrations are essential and synergize with a kinetically distinct second signal. Biochem. J. 322: (Pt. 3):709-718. [Medline]
  28. Dahlgren, C., A. Karlsson, J. Bylund. 2007. Measurement of respiratory burst products generated by professional phagocytes. Methods Mol. Biol. 138: 349-363.
  29. Christophe, T., A. Karlsson, C. Dugave, M. J. Rabiet, F. Boulay, C. Dahlgren. 2001. The synthetic peptide Trp-Lys-Tyr-Met-Val-Met-NH2 specifically activates neutrophils through FPRL1/lipoxin A4 receptors and is an agonist for the orphan monocyte-expressed chemoattractant receptor FPRL2. J. Biol. Chem. 276: 21585-21593. [Abstract/Free Full Text]
  30. Hartt, J. K., G. Barish, P. M. Murphy, J. L. Gao. 1999. N-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two N-formylpeptide receptor (FPR) subtypes: molecular characterization of FPR2, a second mouse neutrophil FPR. J. Exp. Med. 190: 741-747. [Abstract/Free Full Text]
  31. Fu, H., L. Bjorkman, P. Janmey, A. Karlsson, J. Karlsson, C. Movitz, C. Dahlgren. 2004. The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide. BMC Cell. Biol. 5: 50[Medline]
  32. Heit, B., S. Tavener, E. Raharjo, P. Kubes. 2002. An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients. J. Cell Biol. 159: 91-102. [Abstract/Free Full Text]
  33. Fu, H., J. Bylund, A. Karlsson, S. Pellme, C. Dahlgren. 2004. The mechanism for activation of the neutrophil NADPH-oxidase by the peptides formyl-Met-Leu-Phe and Trp-Lys-Tyr-Met-Val-Met differs from that for interleukin-8. Immunology 112: 201-210. [Medline]
  34. Ye, R. D., S. L. Cavanagh, O. Quehenberger, E. R. Prossnitz, C. G. Cochrane. 1992. Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide receptor. Biochem. Biophys. Res. Commun. 184: 582-589. [Medline]
  35. Boulay, F., M. Tardif, L. Brouchon, P. Vignais. 1990. The human N-formylpeptide receptor: characterization of two cDNA isolates and evidence for a new subfamily of G-protein-coupled receptors. Biochemistry 29: 11123-11133. [Medline]
  36. Selvatici, R., S. Falzarano, A. Mollica, S. Spisani. 2006. Signal transduction pathways triggered by selective formylpeptide analogues in human neutrophils. Eur. J. Pharmacol. 534: 1-11. [Medline]
  37. Palmblad, J., C. L. Malmsten, A. M. Uden, O. Radmark, L. Engstedt, B. Samuelsson. 1981. Leukotriene B4 is a potent and stereospecific stimulator of neutrophil chemotaxis and adherence. Blood 58: 658-661. [Abstract/Free Full Text]
  38. Karlsson, J., H. Fu, F. Boulay, C. Dahlgren, K. Hellstrand, C. Movitz. 2005. Neutrophil NADPH-oxidase activation by an annexin AI peptide is transduced by the formyl peptide receptor (FPR), whereas an inhibitory signal is generated independently of the FPR family receptors. J. Leukocyte Biol. 78: 762-771. [Abstract/Free Full Text]
  39. Neumann, J., A. M. Eis-Hubinger, N. Koch. 2003. Herpes simplex virus type 1 targets the MHC class II processing pathway for immune evasion. J. Immunol. 171: 3075-3083. [Abstract/Free Full Text]
  40. Hill, A., P. Jugovic, I. York, G. Russ, J. Bennink, J. Yewdell, H. Ploegh, D. Johnson. 1995. Herpes simplex virus turns off the TAP to evade host immunity. Nature 375: 411-415. [Medline]
  41. Hill, A. B., B. C. Barnett, A. J. McMichael, D. J. McGeoch. 1994. HLA class I molecules are not transported to the cell surface in cells infected with herpes simplex virus types 1 and 2. J. Immunol. 152: 2736-2741. [Abstract]



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