Down-regulation of CXCR1 and CXCR2 expression on human neutrophils upon activation of whole blood by S. aureus is mediated by TNF-α

Abstract

It was suggested that bacterial products can inhibit the expression of leucocyte chemokine receptors during sepsis and affect leucocyte functions in septic syndrome. Superantigens and toxins produced by Staphylococcus aureus are capable of activating leucocytes via binding to MHC-II antigens on monocytes and T-cell receptor molecules on T lymphocytes. It was recently shown that staphylococcal enterotoxins directly down-regulate the expression of CC chemokine receptors on monocytes through binding to MHC class II molecules. We studied the effects of killed S. aureus on the expression of interleukin-8 receptors, CXCR1 and CXCR2, on polymorphonuclear leucocytes (PMN), which are known to lack the expression of MHC-II antigens. It was shown that S. aureus down-regulated the cell-surface expression of CXCR1 and CXCR2 on PMN in the whole blood and total blood leucocyte fraction containing PMN and monocytes, but did not modulate IL-8 receptor expression in purified PMN suspension. Antibody to TNF-α abrogated down-regulation of IL-8 receptors induced by S. aureus. In contrast, LPS reduced CXCR1 and CXCR2 expression in purified PMN and whole blood in a TNF-α-independent manner. We further showed that TNF-α-induced decrease of CXCR1 and CXCR2 expression was associated with lower IL-8 binding and lower CXCR1 and CXCR2 mRNA levels, and was abrogated by protease inhibitors. We suggest that during septicemia, S. aureus may inhibit neutrophil responsiveness to IL-8 and other CXC chemokines via TNF-α- mediated down-regulation of CXCR1 and CXCR2.

Introduction

The response of leucocytes to chemoattractant cytokines (chemokines) is a central event in inflammatory responses. Interleukin-8 (IL-8) belongs to the family of CXC chemokines and was shown to play a central role in recruiting polymorphonuclear leucocytes (PMN) to sites of inflammation caused by bacterial products or aseptic tissue injury [1,2]. IL-8 attracts and activates PMNs via the specific seven-transmembrane domain, G-protein coupled receptors CXCR1 and CXCR2. CXCR1 selectively binds IL-8 with high affinity, whereas the CXCR2 binds to a broad range of CXC chemokines, including IL-8, neutrophil-activating peptide 2 and melanoma growth-stimulating activity [2,3].

Septicemia is a common clinical problem after infection with Gram-positive or Gram-negative bacteria. Lipopolysaccharide (LPS) from Gram-negative bacteria down-regulates IL-8 receptors on PMN, thus inhibiting leucocyte recruitment into the site of infection [4–7]. Staphylococcus aureus is one of the most common Gram-positive pathogens in cases of sepsis [8–10], which often progress to toxic shock syndrome (TSS) and multi-organ dysfunction [9]. Among S. aureus products that contribute to the septic syndrome are superantigens and cytotoxins, interacting with leucocytes via binding to MHC-II antigens on monocytes, and B lymphocytes and TCR molecules on T cells [10–15].

It was shown that CXCR2 expression was down-regulated in patients with sepsis [16]. CXCR1 and CXCR2 were also down-regulated in patients with HIV and pulmonary tuberculosis [17]. Since IL-8 and other chemokines play a central role in regulating leucocytes, down-regulation of chemokine receptors under the influence of bacterial substances and inflammatory cytokines may be relevant to impairment of leucocyte functions during sepsis [18–20]. It was recently shown that staphylococcal superantigens interacting with MHC class II down-modulate CC-chemokine receptors on human monocytes [21]. The effects of S. aureus on the expression of IL-8 receptors on neutrophils have not been explored.

In the present study, we examined the effects of killed S. aureus (SAC) on the expression of the IL-8 receptors CXCR1 and CXCR2 on PMN. Killed S. aureus possess a natural combination of staphylococcal toxins and have been shown to be potent inducers of TNF-α and other cytokines [22,23]. We report here the ability of SAC to down-regulate CXCR1 and CXCR2 expression on PMN in the whole blood and total blood leucocyte fraction containing PMN and monocytes. We demonstrate that the effect of SAC was TNF-α-mediated, since antibody to TNF-α abrogated down-regulation of IL-8 receptors induced by S. aureus. SAC was unable to inhibit the expression of CXCR1 and CXCR2 in purified neutrophils, while LPS down-regulated CXCR1 and CXCR2 in purified PMN fraction in a TNF-α-independent manner. We suggest that during septicemia, S. aureus can inhibit PMN responsiveness to IL-8 and other CXC chemokines via a TNF-α-mediated inhibition of CXCR1 and CXCR2 expression.

Materials and methods

Reagents

The murine hybridoma E3, producing a monoclonal IgG1 antibody against CXCR1, was generated using a fusion protein consisting of the first 30 amino acids of the CXCR1 and glutathione-S-transferase (GST) as an antigen. The antibody E3 recognized N-terminal amino acids 10–12 (WDF) of human CXCR1 (as determined by Andreas Ludwig, Forschungszentrum, Borstel, Germany). Antibody E3 recognized the same epitope as previously-described monoclonal antibodies [24,25], and was able to inhibit the IL-8 binding to CXCR1 (not shown). Monoclonal antibody to CXCR2 [26] was kindly provided by Dr Andreas Ludwig, Zentrum fur Medizin und Biowissenschaften Forschungszentrum, Borstel, Germany.

Neutralizing anti-TNF-α monoclonal antibody 5 N and neutralizing anti-IL-8 monoclonal antibody WS4 were previously generated in our laboratory [27,28].

cDNAs of CXCR1 and CXCR2, and glutathione-S-transferase (GST) fusion protein, containing 30 N-terminal amino acids of CXCR1 were kindly donated by Prof. K. Matsushima, Kanazawa University, Japan. Recombinant IL-8 (72 amino acids) was kindly provided by Prof. K. Matsushima and Prof. S. Ketlinsky, Institute of Highly Pure Biopreparations, St Petersburg, Russia.

Recombinant TNF-α was from Berhinger-Mannheim Biochemicals, Indianapolis, IN.

Inactivated and fixed Staphylococcus aureus Cowan I (SAC) was prepared as described previously [29]. LPS was from Escherichia coli strain 0111:B4 (Sigma, St Louis, MO).

Isolation of leucocytes

Human neutrophils from heparinized blood of healthy volunteers were prepared by standard methods, including dextran sedimentation, Lymphoprep (Nycomed, Oslo, Norway) centrifugation and lysis of residual erythrocytes in double-distilled water [30]. Neutrophils were 94–96% pure and more than 98% viable (according to Giemsa staining and Trypan Blue exclusion test, respectively). The total leucocyte fraction was separated from heparinized blood after dextran sedimentation and lysis of residual erythrocytes [30].

Incubation of leucocytes and whole blood

Purified neutrophils or the total leucocyte fraction were incubated at 37°C in RPMI containing 2% FCS (Hyclone, Logan, UT) at a concentration of 2–3 × 10⁶ cell/ml in polypropylene tubes (Corning, New York, USA). The SAC suspension was added to the leucocyte suspension or to the whole blood at a final concentration of 0·001% v/v [20], and E. coli LPS was used at a concentration of 100 ng/ml which caused maximal down-regulation of CXCR1 and CXCR2 on neutrophils.

TNF-α was added to the leucocyte suspension or whole blood at concentrations ranging from 0·1 to 5 ng/ml. Most experiments employed 1 ng/ml of TNF-α which was sufficient for maximal effects on receptor expression. TNF-α was neutralized with 20 µg/ml of monoclonal antibody 5 N added to cells or whole blood [27,28], and 20 µg/ml of antibody WS4 [24] was added to neutralize endogenous IL-8; this concentration of WS-4 was sufficient to completely block neutrophil binding of ¹²⁵IL-8 at 20 ng/ml (not shown).

Protease inhibitors were used at the following concentrations: 170 µg/ml formylmethylsulphonylfluoride (PMSF) (Sigma), 10 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin (all from Boehringer Mannheim) and 5 mm EDTA.

Flow cytometry analysis

Isolated neutrophils were washed and 5 × 10⁵ cells were suspended in 100 µl of FACS buffer (PBS, 2% BSA, 2% normal rabbit serum (NRS) in PBS) containing 2 µg of monoclonal antibody specific to CXCR1, or 1 µg of monoclonal antibody against CXCR2, for 40 min at 4°C. After washing, the cells in 100 µl were incubated for 30 min at 4°C with 100 µl of FACS buffer, containing FITC-conjugated rabbit anti-mouse IgG (Sigma, St. Louis, MO). Cells were washed twice, fixed and analysed by FACScan (Becton Dickinson, Mountain View, CA) using Lysis II software. Cell staining was also performed with the whole blood. Briefly, whole blood cells were washed twice in PBS, suspended in FACS buffer and processed as described. Erythrocytes were lysed by FACScan lysing-fixing solution (Becton Dickinson).

Determination of cytokines in culture supernatant fluids and plasma

IL-8 and TNF-α were measured by ELISA as described [27,28].

¹²⁵I-IL-8 receptor binding experiments

The chloramine T method was used for IL-8 iodination [31]. Neutrophils (2·5 × 10⁵) were incubated with 10 ng of iodinated IL-8 in 100 µl of PBS containing 2% BSA for 90 min on ice. Cell suspensions were transferred into thin plastic tubes containing 0·4 ml of cold 10% sucrose in PBS, and the cells were separated from the medium by centrifugation for 2 min at 4°C. Non-specific binding was measured after pre-incubation of the cells in the presence of a 200-fold excess of unlabelled IL-8. Tubes were frozen at − 20°C, and cell sediments were sliced off and counted in a γ-counter.

Reverse transcriptase–polymerase chain reaction analysis of purified neutrophil mRNA

Total RNA from neutrophils was extracted by acidic guanidine thiocyanate. Total RNA (5 µg) was reverse-transcribed with M-MLV reverse transcriptase (Perkin Elmer, San Jose, CA), using the recommended conditions, in a total reaction volume of 20 µl containing 1 µg of oligo dT_12-18 (Perkin Elmer).

The products obtained by the reverse transcription were amplified by PCR using the following specific oligo primer pairs:

5′-ATGTCAAATATTACAGATCC-3′— sense CXCR1 (nucleotides 1–20);

5′-AGATTCATAGACAGTCCCCA-3′— antisense CXCR1 (nucleotides 500–481);

5′-GAGGACCCAGGTGATCCAGG-3′— sense CXCR2 (nucleotides 816–835);

5′-GAGAGTAGTGGAAGTGTGCC-3′— antisense CXCR2 (nucleotides 1065–1046).

The anticipated PCR products are 500 bp for CXCR1, 249 bp for CXCR2, 268 bp for β₂-microglobulin (5′-CCAGCAGAGAAT GGAAAGTC-3′— sense β₂-microglobulin, 5′-GATGCTGCTT ACATGTCTCG-3′— antisense β₂-microglobulin). PCR conditions were as follows: melting at 95°C for 1 min, annealing at 60°C and 1 min extention at 72°C for 30 cycles. A total of 10 µl of each final PCR product was size-fractionated in 1·5% agarose gel in the presence of ethidium bromide. Quantification of fluorescence of cellular product bands was performed with Scion Image Software (Frederick, MD, USA). To correct for any variation in RNA content and cDNA synthesis in the different preparations, each sample was normalized on the basis of its β₂-microglobulin content. Results were expressed as the calculated ratio of CXCR mRNAs to β₂-microglobulin mRNA as external control.

Real-time PCR detection of CXCR1, CXCR2 and β-actin mRNA was carried out using the GeneAmp 5700 Sequence Detection System (Perkin-Elmer, PE Biosystems, Warrington, UK). The SYBR Green I PCR Core Reagent kit was used to detect PCR products as SYBRgreen dye is fluorescent when bound to double-stranded DNA. The result of real-time PCR was expressed as the threshold cycle (C_T). The C_T represents the PCR cycle at which the reported fluorescence rises above a set baseline threshold when the DNA amplicon is replicating exponentially. The relative level of CXCR1 and CXCR2 messages was determined by comparing the CXCR1 and CXCR2 to β-actin (5′-TCCTGTGGCATCCACGAAACT- 3′— sense β-actin primer, 5′-GAAGCATTTGCGGTGGACGAT-3′— antisense β-actin primer). In the exponential phase, a C_T difference of 1 is a doubling in the amount of amplicon. Therefore, to determine relative message levels, 2 was raised to the power of ΔC_T (the difference between C_T from treated and untreated cells).

Results

Down-regulation of CXCR1 and CXCR2 expression on human neutrophils in whole blood by SAC or LPS

In septic bacteremia, S. aureus may come into contact with leucocytes in the bloodstream. To examine the effects of S. aureus on the expression of neutrophil IL-8 receptors, fresh blood anticoagulated with heparin was incubated with SAC suspension. CXCR1 and CXCR2 expression on PMN was examined by flow cytometric analysis. SAC significantly reduced the expression of both CXCR1 and CXCR2 on PMN (Fig. 1). Similarly, LPS also decreased the expression of CXCR1 and CXCR2 on PMN. The effect of LPS on CXCR1 and CXCR2 expression was more rapid than the effect of SAC (Fig. 1; similar kinetics were observed in three separate experiments).

Figure 1.  Inhibition of CXCR1 and CXCR2 expression on human neutrophils in whole blood by SAC (a, b) and LPS (c, d). Data from one representative experiment as means of duplicates is shown. The differences between double FACScan estimates were in the range of 2 to 10%. The expression levels of CXCR1 and CXCR2 are displayed as relative units of mean fluorescence intensity (MFI). Anti-TNF-α antibody 5 N was added at 20 µg/ml at the beginning of incubation. Dynamics of TNF-α accumulation during incubation of whole blood with SAC are shown in the frames. (●), None; (▪), SAC; (▴), anti-TNF

Anti-TNF-α blocks the effect of SAC on CXCR1 and CXCR1 expression on human neutrophils in whole blood

We have shown before that SAC is a potent stimulator of TNF-α production in whole blood [22]. Since TNF-α was shown to decrease the expression of CXCR2 on PMN [32], we explored the effects of anti-TNF-α on down-regulation of neutrophil CXCR2 and CXCR1 caused by SAC or LPS in whole blood.

It was shown that neutralization of TNF-α prevented CXCR1 and CXCR2 decrease induced by SAC in whole blood (Figs 1a,b and 2) but had no effect on CXCR down-regulation by LPS (Figs 1c,d and 2) Exogenous TNF-α added to whole blood down-regulated the expression of both receptors. At 2 and 3 h of whole blood incubation with LPS (Fig. 1), anti-TNF-α antibody had little increasing effect on CXCR1 and CXCR2 expression (similar results were obtained in five independent experiments). Anti-TNF-α antibody alone had no effect on CXCR1 and CXCR2 expression (not shown). Both SAC and LPS induced accumulation of TNF-α in the blood during a 3 h incubation (Fig. 1), and the average levels of TNF-α induced by SAC and LPS were similar in seven independent experiments (SAC induced 1·4 ± 1·34 ng/ml of TNF-α and LPS induced 2·2 ± 1·03 ng/ml).

Figure 2.  Effect of SAC, LPS and TNF-α on CXCR2 and CXCR1 expression on neutrophils during 2 h incubation of whole blood. Data from four experiments are presented as percentages of remaining fluorescence on treated cells compared with untreated incubated controls (100%) (mean ± s.e.m.). Anti-TNF-α antibody 5 N and anti-IL-8 antibody WS-4 were added at 20 µg/ml at the beginning of incubation. *P < 0·05 versus untreated incubated controls, **P < 0·05 versus SAC. The statistical differences between groups were determined by Student's t-test. A value of P < 0·05 was considered as significant.

Neutralization of endogenous IL-8 had no effect on CXCR1 and CXCR2 expression on human neutrophils in whole blood

It was previously shown that stimulation of whole blood by SAC results in production of IL-8 [23,33]. Since IL-8 at a concentration of about 50–100 ng/ml may induce internalization of CXCR1 and CXCR2 [34], we explored the role of endogenous IL-8 in down-regulation of CXCR2 and CXCR1 caused by SAC and LPS. Stimulation of whole blood with SAC and LPS in the present experiments resulted in accumulation of 2·6 ± 1·4 ng/ml and 1·8 ± 1·2 ng/ml of IL-8 protein, respectively, at the 2 h point of whole blood incubation (mean of seven independent experiments). These concentrations are less than that required for internalization of IL-8 receptors [34]. Nevertheless, we explored the effect of anti-IL-8 on down-regulation of CXCR2 and CXCR1. Figure 2 shows that adding the neutralizing anti-IL-8 monoclonal antibody WS4 to whole blood did not prevent down-modulation of CXCR1 or CXCR2 on PMN after either SAC or LPS stimulation.

Staphylococcus aureus is unable to down-regulate CXCR1 and CXCR2 expression on purified human neutrophils

Since it was shown that endogenous TNF-α plays an obligatory role in SAC-mediated down-regulation of the expression of CXCR1 and CXCR2 on PMNs in whole blood, we explored the effect of SAC on purified human PMNs. Figure 3 shows that SAC is unable to cause any effect on the expression of CXCR1 and CXCR2 on purified PMNs; similarly, we detected no TNF-α production in purified neutrophils incubated with SAC for 2·0 and 24 h (not shown). Exogenous TNF-α at a concentration of 1 ng/ml decreased the expression of both CXCR1 and, particularly, CXCR2 on PMNs during 2·0 h of cell incubation; the effect was abrogated by anti-TNF-α antibody.

Figure 3.  The effect of SAC on CXCR1 and CXCR2 expression on purified human neutrophils (a, b) and on neutrophils of total blood leucocyte fraction (c, d). Purified neutrophils or total leucocytes at 2 × 10⁶ were incubated with SAC (0·001% v/v) for 2 h. The expression of CXCR1 and CXCR2 is shown as relative units of mean fluorescence intensity (MFI). Anti-TNF-α or anti-IL-8 antibody was added at 20 ng/ml at the beginning of incubation. Mean of duplicates is shown, and the difference between double FACScan estimates was in the range of 2 to 10%. Similar results obtained in two independent experiments with total leucocyte fraction, and five experiments with purified neutrophils.

Staphylococcal antigens have been shown to directly activate monocytes, B and T lymphocytes via binding to MHC-II antigens on monocytes, and B cells and TCR molecules of T cells [10–15]. We explored the effect of SAC on the CXCR neutrophil expression in the total blood leucocyte fraction containing monocytes, lymphocytes and granulocytes. In contrast to purified PMNs, the total peripheral blood leucocyte fraction (Fig. 3) responded to SAC with down-regulation of CXCR1 and CXCR2 on neutrophils. Similar to the effect of SAC on whole blood, down-regulation of IL-8 receptors caused by SAC in the total leucocyte fraction was abrogated by anti-TNF-α antibody (Fig. 3). It was noteworthy that total leucocytes responded to SAC with production of TNF-α at 0·5–2·0 ng/ml. Anti-IL-8 antibody had no influence on inhibition of CXCR1 and CXCR2 expression on PMN caused by SAC in the total leucocyte fraction (Fig. 3).

Exogenous TNF-α down regulates CXCR1 and CXCR2 expression in human neutrophils from whole blood and in purified neutrophils

It was previously shown [29] that TNF-α decreased the surface expression of CXCR2, but not CXCR1, during 30 min of PMN incubation. In our study, SAC exerted TNF-α-mediated down-regulation of both CXCR1 and CXCR2 expression on neutrophils. To explore whether TNF-α is capable of down-regulating the expression of both CXCR1 and CXCR2 on PMN, we incubated purified neutrophils with recombinant TNF-α. The addition of TNF-α to purified neutrophil suspensions caused rapid and dose-dependent down-regulation of CXCR1 and CXCR2 expression (Fig. 4), and drastic reduction of iodinated IL-8 binding (Fig. 4). After 2 h of purified neutrophil incubation, TNF-α reduced CXCR1 receptor expression by 15–30%, CXCR2 by 25–50% and iodinated IL-8 binding by 65% in a dose-dependent manner. The maximum effect was observed with 1 ng/ml TNF-α(Fig. 4). The effect was eliminated by pre-incubating TNF-α with neutralizing monoclonal antibody 5 N (not shown). Reduction of CXCR1 and CXCR2 expression by TNF-α was time-dependent, but the kinetics of CXCR1 and CXCR2 down-regulation were different. CXCR2 was found to be more susceptible to TNF-α action at earlier time points, 0·5 and 1 h (Fig. 5).

Figure 4.  (a) Dose-dependent effect of TNF-α on I¹²⁵-labelled IL-8 binding to human neutrophils. Neutrophils were incubated with TNF-α for 2 h. Results of one representative experiment (n = 4) is shown (similar results obtained in three separate experiments). Data are presented as Mean ± SD. (b) Dose-dependent effect of TNF-α on the expression of CXCR1 and CXCR2 on human neutrophils. Neutrophils were incubated with different concentrations of TNF-α for 2 h. The expression levels of CXCR1 and CXCR2 are displayed as relative units of fluorescence intensity (MFI). Data of one representative experiment are shown as the arithmetic means of duplicate determinations (similar results obtained in three separate experiments). (●), CXCR1; (▪), CXCR2.

Figure 5.  Differential kinetics of (a) CXCR1 and (b) CXCR2 down-modulation on human neutrophils, incubated in the presence of TNF-α. Data of one representative experiment (means of triplicates) are shown. The expression levels of CXCR1 and CXCR2 are displayed as relative units of mean fluorescence intensity. (○), Neutrophils, incubated without TNF-α; (▴), neutrophils, incubated in the presence of 1 ng/ml TNF-α.

The effect of TNF-α on purified human neutrophils is reduced by protease inhibitors

The addition of a mixture of protease inhibitors (leupeptin, pepstatin, aprotinin and EDTA) and TNF-α to the culture medium sustained the normal level of binding for anti-CXCR1 and anti-CXCR2 monoclonal antibodies on PMN. Surprisingly, TNF-α-induced reduction of the iodinated IL-8 binding was not restored by protease inhibitors (Table 1).

Table 1.   Down-regulation of CXCR1 and CXCR2 receptor expression on human neutrophils after 2 h incubation of purified neutrophils with 1 ng/ml of TNF-α

	None	TNF-α	TNF-α+ anti-TNF-α	TNF-α+ protease inhibitors
Anti-CXCR1 and anti-CXCR2 antibody binding to the cells was assessed by FACS and presented as mean fluorescence intensity (MFI) of duplicates. The overall binding of iodinated IL-8 to neutrophils is presented in c.p.m. (mean ±s.d. of triplicates). Similar data obtained in three independent experiments.
CXCR1, MFI	125	78	125	124
CXCR2, MFI	73	44	75	61
I^125IL-8 binding, c.p.m.	8847 ± 303	3613 ± 392	8050 ± 15	3659 ± 352

TNF-α down regulates CXCR1 and CXCR2 mRNA expression in purified human neutrophils

TNF-α reduced the CXCR1 and CXCR2 mRNA content in human neutrophils incubated for 120 min (Fig. 6), while we detected no CXCR1 and CXCR2 mRNA down-regulation at the 30 min and 60 min time points (not shown). Similarly to the data of Fig. 6, real-time PCR detection of CXCR1, CXCR2 and β-actin mRNA revealed that the CXCR1 mRNA content was reduced to less than 10%, and CXCR2 mRNA content was reduced to less than 20% of control cells at 2 h of incubation (not shown).

Figure 6.  Down-regulation of CXCR1 (□) and CXCR2 (▪) mRNA expression in human neutrophils at 120 min of incubation. Cells were incubated for 30 min, 60 min and 120 min in the presence or absence of TNF-α alone or TNF-α pre-incubated with neutralizing anti-TNF-α antibody 5 N. No down-regulation of CXCR1 or CXCR2 mRNA was detected at 30 min and 60 min compared with the control level without TNF-α (not shown). Similar results were obtained in five separate experiments. PCR analysis of mRNA as presented above was used in three experiments and real-time PCR was used in two experiments (not shown).

Discussion

Down-regulation of leucocyte chemokine receptors and chemokine responsiveness under the influence of bacterial substances and inflammatory cytokines may be relevant to the impairment of antibacterial functions of leucocytes during sepsis [18–20]. Here, we demonstrate that Gram-positive bacteria S. aureus have the capacity to down-regulate the expression of IL-8 receptors on human neutrophils.

Staphylococcal superantigens can activate MHC-II-rich leucocyte subsets, monocytes and B lymphocytes by binding directly to MHC class II molecules, and can activate T cells by co-engagement of both MHC class II and TCR [13–15,21,35]. The pathological sequences of these processes are derived from an enormous secretion of proinflammatory cytokines, including TNF-α and IL-1 [21]. It was recently shown that staphylococcal superantigens down-modulate the expression of CC chemokine receptors in human monocytes through MHC-II antigen-associated cellular tyrosine protein kinases; the effect was independent of soluble cytokine production[21].

PMN do not normally express MHC class II molecules [35,36] and therefore, are not believed to interact directly with staphylococcal antigens. In our study, addition of SAC to human blood or the total leucocyte fraction containing monocytes and lymphocytes down-regulated the expression of both CXCR1 and CXCR2 on neutrophils. In contrast, SAC did not affect the expression of IL-8 receptors in purified neutrophils, suggesting a critical role of other leucocyte subsets, monocytes and lymphocytes in this process. SAC has been shown previously to be a potent inducer of TNF-α and other cytokines in whole blood and monocyte cultures [23,37]. Our data demonstrate that SAC-induced down-regulation of CXCR1 and CXCR2 on neutrophils in whole blood and the total leucocyte fraction was abrogated by neutralizing antibody to TNF-α, indicating that the effect was indirect and mediated via TNF-α produced by other leucocyte subsets. These data are in agreement with the recent demonstration that the effects of staphylococcal toxins on neutrophil respiratory burst activity and apoptosis are indirect and completely mediated via monocyte and T-cell-derived cytokines, while purified neutrophils do not respond to the toxins [38]. It seems likely that isolated staphylococcal toxins [36,38], as well as killed SAC bacteria exposing a spectrum of cell-wall staphylococcal toxins, have no direct effect on human neutrophils which do not express MHC-II antigens. It is worth noting that neutrophils have been shown recently to express MHC II molecules and to interact with staphylococcal superantigens when cultured with GM-CSF or interferon gamma [35,36], suggesting that in some circumstances, staphylococcal toxins can directly affect neutrophils via MHC II binding.

The indirect effect of SAC on neutrophils contrasts with the effect of LPS from Gram-negative bacteria that modulated the expression of IL-8 receptors in a TNF-α-independent manner. In addition, the effect of LPS occurred faster compared with the effect of SAC, which was secondary to accumulation of TNF-α. It is noteworthy that although LPS induced substantial TNF-α production in the whole blood and total leucocytes, it seems that TNF-α had no impact on LPS-induced down-regulation of CXCR, since it was resistant to anti-TNF-α antibody.

Our observation on TNF-α-mediated down-regulation of both CXCR1 and CXCR2 on neutrophils seems to be in conflict with a previous demonstration of CXCR2, but not CXCR1, expression inhibition by TNF-α during a 30 min exposure to cytokine [32]. In our study, neutrophils were exposed to TNF-α for a longer period and this may explain the down-regulation of both CXCR1 and CXCR2, since a decrease of CXCR1 expression was observed later compared with CXCR2.

It was recently demonstrated that proteases may be involved in CXCR1 and CXCR2 down-regulation by TNF-α[4,5]. In our experiments, the addition of a mixture of protease inhibitors prevented TNF-α-dependent decrease of anti-CXCR1 and anti-CXCR2, as assessed by FACS analysis of anti-CXCR monoclonal antibodies binding to neutrophils. Surprisingly, TNF-α-mediated reduction of the binding of iodinated IL-8 was not restored by protease inhibitors. The discrepancy between the levels of FACS staining and IL-8 binding is probably due to incomplete inhibition of receptor proteolytic degradation when N-terminal extracellular portions of receptors were still recognized by anti-CXCR antibodies, while ligand binding requiring interaction with a few sites on the receptor molecule [39] was not restored. The differential kinetics of TNF-α-induced CXCR1 and CXCR2 degradation shown herein may be due to differential susceptibility of CXCR1 and CXCR2 to proteolysis bound to different rates of CXCR1 and CXCR2 protein glycosylation. Although CXCR1 and CXCR2 molecular masses, deduced from cDNA sequences, are about 40 kDa [40], and neutrophil membrane CXCR2 is the 45 kDa receptor protein [30], we have shown recently that the main form of cell surface CXCR1 is represented as a 70 kDa protein [41], implying a high level of CXCR1 glycosylation.

Besides proteolytic CXCRs reduction, we have shown that TNF-α decreased CXCR1 and CXCR2 mRNA levels in PMN, and this may partially account for the change in receptor proteins levels observed at later time points. However, proteolytic degradation is the most rapid and probably the most effective way of modulating IL-8 receptors on PMN.

Recently, it was shown that CXCR2 expression on neutrophils is decreased in patients with sepsis [16], although possible mechanisms were not identified. Our observations on TNF-α-mediated down-regulation of CXCR1 and CXCR2 by staphylococcal bacteria suggest that this mechanism may take part in the pathogenesis of sepsis and local infections caused by S. aureus. In the case of sepsis, the lack of IL-8 receptor expression may sequester the activated neutrophils in the bloodstream, thereby contributing to severe complications of septicemia. Our data, together with the demonstration of TNF-α-mediated induction of acute inflammation in vivo and TNF-α-mediated injury of endothelial cells by staphylococcal toxins [15,42], further reveal the critical role of TNF-α in pathological perturbations caused by Gram-positive staphylococci.

Ackknowledgements

We thank Natalia Nashkevich and Svetlana Koleda (Inst. Hematology, Minsk, Belarus) for kind assistance with cytokine ELISAs, Dr Andres Ludwig (Forschungszentrum, Borstel, Germany) for kind donation of antibody to CXCR2, Prof. Kougi Matsushima (Kanazawa University, Japan) for donation of glutathione-S-transferase (GST)-N-terminus CXCR1 fusion protein and recombinant IL-8, Prof. Sergei Ketlinsky (Institute of Highly Pure Biopreparations, St Petersburg, Russia) for the gift of recombinant IL-8 and Douglas Horejsh (Department of Pathology, University of Wisconsin) for his guidance in real-time PCR detection of CXCR1 and CXCR2 mRNA. The work was supported by Grant 94-1634 from INTAS, Brussels, research funding from the Office of International Affairs, Department of Health & Human Services, National Cancer Institute, NIH, Bethesda, Maryland, Grants from Fund for Fundamental Investigations of Belarus and Belorussian Ministry of Health, and USPHS grant AI/HD 36643 (C.D.P.).

Footnotes

1. I. Tikhonov and T. Doroshenko contributed equally to this study