Inhibition of interleukin-8 production in human endothelial cells by Staphylococcus aureus supernatant

Abstract

Recent reports have shown that Staphylococcus aureus infection increases the expression of cytokines and cell adhesion molecules in endothelial cells and enhances leucocyte migration, thereby resulting in bacterial elimination. In this study, we analysed the production of the chemokine interleukin (IL)-8 in human umbilical vein endothelial cells (HUVEC) infected with several S. aureus strains by using reverse transcription–polymerase chain reaction and enzyme-linked immunosorbent assay. We found that the avirulent strains (00–51 and 00–62) increased IL-8 production but the virulent strains (A17 and A151) decreased it at both the mRNA and protein levels. We considered that the inhibition of IL-8 production depended on certain inhibitory factor(s) secreted by bacteria. This was because S. aureus also abolished IL-8 expression in HUVEC treated with cytochalasin D, and the addition of culture supernatants of strains A17 and A151 decreased IL-8 production in HUVEC. This factor(s) in the bacterial culture supernatant inhibited both basal and tumour necrosis factor (TNF)-α-induced IL-8 production. In contrast, no inhibitory effect was observed on monocyte chemotactic protein-1 (MCP-1) production. These results indicate that S. aureus can down-regulate IL-8 release in endothelial cells through the secretion of inhibitory factor(s), and this may result in decreased neutrophil recruitment, thus interfering with the host immune response to bacterial infection.

Introduction

Staphylococcus aureus is a Gram-positive pathogen that colonizes the anterior nares of 30% to 50% of healthy adults; of these, with 20% are persistently colonized. This organism can cause various infections, ranging from superficial ones such as abscesses and other skin infections to serious invasive infections such as sepsis and endocarditis [1]. Moreover, S. aureus is a leading cause of nosocomial infections, and its colonization is a known risk factor for invasive diseases.

The innate immune system plays a crucial role in the host response to S. aureus infection [2]. As an immediate host response toward bacterial infection, leucocytes migrate from the circulation to the infection site. The endothelium plays an important role in neutrophil recruitment by modulating the expression of cell adhesion molecules such as E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cellular adhesion molecule (VCAM)-1, as well as cytokines such as interleukin (IL)-8 [3]. IL-8 is synthesized by endothelial cells in response to inflammatory signals such as tumour necrosis factor (TNF)-α and IL-1β; it is a potent neutrophil chemoattractant and promotes neutrophil transendothelial migration [4]. Recently, it has been reported that some infectious pathogens induced the expression of chemokines and adhesion molecules in human endothelial cells [5–11]. S. aureus is one such pathogen, and several studies have shown that when internalized by endothelial cells, S. aureus induced the up-regulation of ICAM-1 and the secretion of cytokines, including IL-8 in these cells [5–8]. This activation of endothelial cells leads to leucocyte recruitment. Thus, it is suggested that S. aureus-infected endothelial cells may develop a mechanism for bacterial elimination through chemoattractant production and neutrophil recruitment.

The present study was conducted to determine whether S. aureus strains vary in their ability to induce IL-8 production in endothelial cells and whether this ability is correlated with bacterial virulence. We investigated IL-8 production in endothelial cells in response to several avirulent and virulent S. aureus strains in a mouse infection model, and we analysed the relationship between IL-8 induction and bacterial virulence.

Materials and methods

Reagents

Recombinant human TNF-α was obtained from R&D Systems (Wiesbaden, Germany). Cytochalasin D was purchased from Sigma (St Louis, MO, USA).

Endothelial cell cultures

Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Walkersville, MD, USA) and grown in 25-cm tissue culture flasks coated with 0·2% gelatin (Sigma) in HEC-C1 medium which consisted of a medium MCDB107, acid fibroblast growth factor and heparin (IFP, Yamagata, Japan) containing 10% fetal bovine serum (FBS) and supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were cultured at 37°C in 5% CO₂. Confluent HUVEC were treated with trypsin (0·05%)-ethylenediamine tetraacetic acid (EDTA) (0·02%), transferred to new flasks, 35-mm dishes or 24-well plates and used between three and six passages.

Bacterial cell cultures

We used two clinical isolates (A17 and A151) of S. aureus from atopic skin and two commensal strains (00–51 and 00–62) isolated from healthy nasal carriers. In the mouse abscess model, the former two virulent strains showed higher in vivo survival [the viable count of bacteria from tissues was 0·5–1 × 10⁹ colony-forming units (CFU)] than the latter avirulent strains (the viable count of bacteria from tissues was 2·5 × 10⁵ CFU). The bacteria were cultured overnight in brain heart infusion (BHI) broth, diluted 60-fold in fresh BHI broth, and grown to mid-log phase. The cultured bacteria were harvested by centrifugation, washed with saline and suspended in HEC-C1 without antibiotics at a density of 2·5 × 10⁶ CFU/ml.

Incubation of S. aureus with HUVEC

HUVEC grown to confluence in 24-well plates were washed once with HEC-C1 without antibiotics, and 0·2 ml of each bacterial suspension was added to each well. After 1-h incubation, the cells were washed with the culture medium and incubated in HEC-C1 containing 10 μg/ml lysostaphin to lyse any extracellular bacteria. At different time-points, supernatants were collected and stored at −20°C for cytokine measurement. In some experiments, HUVEC were incubated with supernatant obtained from the 1-h HEC-C1 culture suspension of bacteria that had been passed through a 0·22-μm filter.

To examine the efficiency with which S. aureus infected endothelial cells, the monolayer of infected endothelial cells was fixed with methanol and stained with Giemsa dye. The number of endothelial cells that had internalized one or more bacterial cells as well as the mean number of internalized bacterial cells per infected endothelial cell was determined by light microscopic examination.

Analysis of cell viability and apoptosis

HUVEC were infected with S. aureus as described above and incubated for 24 h. After incubation, the cells were collected and analysed for viability and apoptosis. Cell viability was measured by the trypan blue dye exclusion method, and apoptosis was measured by annexin V staining. For the apoptosis analysis the cells were incubated with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), according to the manufacturer's instructions (annexin V-FITC kit; Beckman Coulter, Inc., Fullerton, CA, USA). The stained cells were analysed by flow cytometry (FACSCalibur; BD Biosciences Clontech, Palo Alto, CA, USA) to determine the percentage of annexin V-positive/PI-negative cells (apoptosis) or annexin V/PI double-positive cells (necrosis). Data acquisition (10⁴ events per sample) and analysis were carried out using the CellQuest software (BD Biosciences).

Enzyme-linked immunosorbent assay (ELISA) for IL-8 and monocyte chemotactic protein-1 (MCP-1)

IL-8 and MCP-1 in the supernatant of endothelial cells were quantified by using an ELISA kit (BioSource International, Inc., Camarillo, CA, USA) according to the manufacturer's instructions.

Reverse transcription–polymerase chain reaction (RT–PCR)

HUVEC cultured in a 35-mm dish was incubated with 1 ml of bacterial suspension for 1 h and cultured as described above; they were then harvested with Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) to isolate total cellular RNA. Total RNA was isolated and treated with RNase-free Dnase 1 (Invitrogen, Karlsruhe, Germany), according to the manufacturer's instructions. DNase-treated total RNA was reverse transcribed at 50°C for 1 h using the one-step RT–PCR kit (BD Biosciences Clontech). The RT reaction products were subsequently PCR-amplified in a thermal cycler (Techgene; Techne, Cambridge, UK) by using the following primers: IL-8: forward, 5′-ATGACTTCCAAGCTGGCCGTGGCT-3′ and reverse, 5′-TCTCAGCCCTCTTCAAAAACTTCTC-3′; and β-actin: forward, 5′-GTGGGGCGCCCCAGGCACCA-3′ and reverse, 5′-CTCCTTAATGTCACGCACGATTTC-3′. The PCR conditions were as follows: initial denaturation at 94°C for 5 min and 94°C for 30 s, annealing at 65°C for 30 s, 1 min at 68°C for 28 cycles and final extension at 68°C for 2 min. The PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining.

Statistical analyses

Results are expressed as mean ± standard deviation. The significance of difference between the mean results for the different groups was determined by Student's t-test. A P-value < 0·05 was considered statistically significant.

Result

IL-8 production in S. aureus-infected endothelial cells

Culture supernatants derived from uninfected or S. aureus-infected endothelial cells were analysed by ELISA for the presence of IL-8. Uninfected HUVEC cultures were found to constitutively express low levels of IL-8 (Fig. 1a). IL-8 production was induced in HUVEC infected with the avirulent strains 00–51 and 00–62 after 24 h of infection. At both 6 and 24 h, IL-8 production in HUVEC infected with the virulent S. aureus strains A17 and A151 was reduced by 30–50% compared to that in uninfected HUVEC (Fig. 1a).

Fig. 1.

Effect of Staphylococcus aureus on interleukin (IL)-8 production in endothelial cells. Human umbilical vein endothelial cells (HUVEC) were co-cultured with avirulent (00–51 and 00–62) or virulent (A17 and A151) strains for 1 h, washed and incubated in medium containing lysostaphin to lyse any extracellular bacteria. (a) IL-8 secretion was analysed by enzyme-linked immunosorbent assay (ELISA) in the cell culture supernatants collected at 6 and 24 h after infection. The data are represented as means ± standard deviation for at least three separate experiments. *P-value of < 0·01 compared to uninfected HUVEC. (b) After infection, HUVEC were treated with lysostaphin for 30 min, fixed and stained. The mean number of intracellular bacterial cells per infected endothelial cell was determined. The data are represented as means ± standard deviation for at least three separate experiments.

Several studies demonstrated that the induction of cytokine expression in S. aureus-infected endothelial cells required internalization of bacteria, and S. aureus strains that exhibited a low internalization rate did not induce cytokine expression [5,7]. The invasion frequencies and the mean number of internalized bacterial cells were analysed. The percentage of infected HUVEC for each of the four test strains was almost 100% (data not shown), and approximately 20 intracellular bacterial cells per infected endothelial cell were observed (Fig. 1b). Furthermore, no major difference was observed in the average number of intracellular bacterial cells after 6 and 24 h of incubation (data not shown). The strains tested showed no difference with regard to the number of bacteria internalized and the frequency of internalization.

To exclude the possibility that the infection of endothelial cells with S. aureus resulted in cell damage and a reduction in IL-8 production, we tested cell viability by the trypan blue dye exclusion method at 24 h after infection. The cell viability of S. aureus-infected HUVEC was similar to that of uninfected cells (Table 1). We also tested the binding of annexin V to HUVEC after 24 h of infection in order to investigate whether apoptosis contributes to a decrease in IL-8 production in HUVEC. The percentage of apoptotic cells (annexin-positive/PI-negative) increased by treatment with cycloheximide and TNF but not by bacterial infection. Similarly, no difference was observed in the percentage of necrotic cells (annexin/PI double-positive) between the uninfected and infected HUVEC. Obvious cytotoxic effects were not observed in HUVEC infected with the test bacterial strains (Table 1). These results indicate that the inhibition of IL-8 production in S. aureus-infected HUVEC was not a result of cell damage.

Table 1.

Effect of Staphylococcus aureus on cell viability and apoptosis.

Treatmenta	Cell viability (%)b	Annexin V (+), PI (–) cells (%)c	Annexin V (+), PI (+) cells (%)c
Uninfected	96·9 ± 1·27	6·87	2·51
A17	96·0 ± 1·34	5·89	2·23
A151	95·9 ± 1·98	7·17	3·74
00–51	97·4 ± 1·7	5·14	2·84
00–62	97·7 ± 0·35	4·28	2·90
TNF/CHX	–	12·8	3·34

^aHuman umbilical vein endothelial cells (HUVEC) were infected with each S. aureus strain and harvested 24 h after infection.

^bCell viability was assessed by the trypan blue exclusion method. The cells were treated with trypan blue dye, and the number of viable cells was determined. The data are represented as means ± standard deviation for at least three separate experiments.

^cApoptosis was measured by annexin V staining. Endothelial cells were stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for flow cytometric analysis, and the percentage of annexin V-positive/PI-negative cells or annexin V/PI double-positive cells were evaluated. As a positive control for apoptosis, HUVEC were treated with 10 μg/ml cycloheximide (CHX) and 30 ng/ml TNF-α for 4 h.

Infection of HUVEC with S. aureus reduced IL-8 mRNA level

To determine whether infection of HUVEC with S. aureus inhibited the production of IL-8 mRNA, RNA was extracted from S. aureus-infected endothelial cells at 3 and 12 h after infection, and the IL-8 mRNA level was analysed by RT–PCR. At 12 h after infection, the IL-8 mRNA level increased in HUVEC co-cultured with strains 00–51 and 00–62 compared to that in uninfected HUVEC. However, in S. aureus A17- and A151-infected HUVEC, we observed a reduction in the IL-8 mRNA levels at both 3 and 12 h after infection (Fig. 2).

Fig. 2.

Effect of Staphylococcus aureus on interleukin (IL)-8 mRNA expression in endothelial cells. Human umbilical vein endothelial cells (HUVEC) monolayers were infected with each S. aureus strain. Total RNA was then extracted at 3 and 12 h post-infection and subjected to reverse transcription–polymerase chain reaction (RT–PCR). The PCR products were analysed on a 2% agarose gel and visualized by ethidium bromide staining. Oligonucleotides specific to β-actin were used as house-keeping genes. UI: uninfected endothelial cells.

Bacterial culture supernatants of S. aureus strains inhibit IL-8 production

To elucidate whether the internalization of bacteria was required for the inhibition of IL-8 production, HUVEC were treated with cytochalasin D − an inhibitor of endocytosis − and infected with the bacteria for 1 h. After 3 h, RNA was extracted from the endothelial cells, and the IL-8 mRNA level was examined by RT–PCR. HUVEC cocultured with S. aureus A17 in the presence of cytochalasin D (1 μg/ml) showed a reduction in the IL-8 gene expression (Fig. 3). The internalization of S. aureus by HUVEC may not be required for the inhibition of IL-8 production, as observed in the case of strain A17.

Fig. 3.

Effect of bacterial invasion on interleukin (IL-8) expression in human umbilical vein endothelial cells (HUVEC). To examine the effect of bacterial invasion on IL-8 expression, HUVEC were pretreated with or without cytochalasin D (1 μg/ml) for 30 min and infected with Staphylococcus aureus A17 or 00–62 strain. The IL-8 mRNA level at 3 h was analysed by reverse transcription–polymerase chain reaction (RT–PCR).

Next, we examined the effect of bacterial culture supernatant on the reduction of IL-8. For 3 h or more, HUVEC were cultured with the supernatant obtained from a 1-h culture of each test strain (2·5 × 10⁶ CFU/ml), and the IL-8 gene expression and protein secretion were analysed. Compared to strains 00–51 and 00–62, the culture supernatants of strains A17 and A151 inhibited IL-8 gene expression (Fig. 4a). Similarly, IL-8 protein secretion was reduced significantly (P < 0·01) by the bacterial culture supernatants of strains A17 and A151 (Fig. 4b). The inhibitory effect was detected at 3 h, and persisted for 24 h. On the other hand, the culture supernatants of the 1-h cultures of strains 00–51 and 00–62 did not affect IL-8 production. These results indicate that the virulent S. aureus strains A17 and A151 secrete certain factor(s) that can decrease IL-8 production in endothelial cells.

Fig. 4.

Effect of bacterial culture supernatant on interleukin (IL)-8 expression. Bacterial culture supernatant prepared from a 1-h culture of each Staphylococcus aureus strain [2·5 × 10⁶ colony-forming units (CFU)/ml] was added to human umbilical vein endothelial cells (HUVEC). IL-8 expression was analysed by reverse transcription–polymerase chain reaction (RT–PCR) after a 3-h incubation (a) and by enzyme-linked immunosorbent assay (ELISA) after 3-, 6- and 24-h incubations (b). The data are represented as means ± standard deviation for at least three separate experiments. *P-value < 0·01 compared to untreated HUVEC (control).

Kinetic analysis of the bacterial culture supernatant

We conducted more detailed study of the inhibitory effect of supernatant obtained from a 1-h culture of strain A17 on IL-8 production in HUVEC. HUVEC were incubated with various dilutions of bacterial culture supernatant with a density of 2·5 × 10⁹ CFU/ml (= 100% supernatant) for 3 h, and were analysed for IL-8 production by ELISA. IL-8 production was reduced when HUVEC were incubated with 0·1–100% dilution of the bacterial culture supernatant, and the maximum inhibition was observed at 1% dilution of the culture supernatant (Fig. 5a). This concentration was used in all subsequent experiments. The inhibitory effect induced by this treatment persisted for 24 h, and it was unlikely that direct toxic effects were induced since the cell viability remained unaffected following 24 h of treatment (data not shown).

Fig. 5.

Analysis of the inhibition of interleukin (IL)-8 production by the bacterial culture supernatant. (a) Bacterial culture supernatant of Staphylococcus aureus A17 [2·5 × 10⁹ colony-forming units (CFU)/ml] was diluted and added to human umbilical vein endothelial cells (HUVEC) at various concentrations (v/v). After a 3-h incubation, IL-8 release was measured by enzyme-linked immunosorbent assay (ELISA). (b) Bacterial culture supernatant was treated with 1 mg/ml of proteinase K or trypsin for 2 h at 37°C or heated at 100°C for 5 and 15 min and then added to HUVEC at 1% (v/v) concentration. After 3 h, supernatants were collected from the HUVEC cultures and analysed for IL-8 production by ELISA. The data are represented as means ± standard deviation for at least three separate experiments. *P-value < 0·01 compared to untreated HUVEC (control).

Bacterial culture supernatant subjected to protease or heat treatment was added to HUVEC at a 1% concentration, and IL-8 secretion was determined after 3 h of incubation. The reduction in IL-8 production was not affected by heating the supernatant for 5 and 15 min at 100°C. Treatment of the supernatant with 1 mg/ml of proteinase K or trypsin at 37°C for 2 h decreased the effect of supernatant on IL-8 production in HUVEC (Fig. 5b). These results indicate that the inhibitory activity for IL-8 expression in the culture supernatant was involved in heat-stable protein.

S. aureus culture supernatants suppress IL-8 but not MCP-1 production in endothelial cells activated by TNF-α

The above results indicate that the bacterial culture supernatant abolished IL-8 production in unstimulated HUVEC. To determine whether the supernatant could inhibit IL-8 production in stimulated endothelial cells, we pretreated HUVEC for 3 h with or without 1% dilution of bacterial culture supernatant that was obtained from 1-h culture, and we then exposed the cells to various concentrations of TNF-α for 6 h. An increase in the TNF-α concentration resulted in increased IL-8 production. Pretreatment with bacterial culture supernatant significantly (P < 0·01) inhibited TNF-α-induced IL-8 production (Fig. 6a).

Fig. 6.

Effect of bacterial culture supernatant on tumour necrosis factor (TNF)-α-induced interleukin (IL)-8 and monocyte chemotactic protein-1 (MCP-1) production in human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with 1% (v/v) of bacterial culture supernatant (sup) for 3 h and stimulated with TNF-α at the indicated concentrations. After 6 h, the supernatants collected from the HUVEC cultures were analysed by enzyme-linked immunosorbent assay (ELISA) for IL-8 (a) and MCP-1 (b). The data are represented as means ± standard deviation for at least three separate experiments. *P-value < 0·01 compared to TNF-α-treated HUVEC. #P-value < 0·05 compared to TNF-α-treated HUVEC.

The production of MCP-1, which is a member of the CC chemokine family and a chemoattractant for monocytes, is induced in endothelial cells following S. aureus infection [12]. To determine whether bacterial culture supernatant also inhibits MCP-1 expression, we analysed MCP-1 production in HUVEC as described above. Unstimulated HUVEC cultures were found to express constitutively low levels of MCP-1. TNF-α induced the release of a significant amount of MCP-1 (Fig. 6b). However, compared to IL-8, the inhibitory effect of bacterial culture supernatant on basal MCP-1 expression was low. Moreover, MCP-1 production increased in the presence of TNF-α (0·1 and 1 ng/ml).

Thus, it was suggested that the bacterial culture supernatant does not inhibited the expression of all chemokines, and its effect is specific to certain genes.

Discussion

Previous studies have revealed that S. aureus-infected endothelial cells express cell adhesion molecules and chemokines such as IL-8. In this study, we demonstrated that cytokine IL-8 production in HUVEC varies according to the virulence of the infecting S. aureus strains. Infection of HUVEC with the avirulent strains (00–51 and 00–62) increased IL-8 expression. On the other hand, IL-8 production in HUVEC infected with the virulent strains (A17 and A151) was reduced at both the protein and mRNA levels. Non-invasive bacteria are unable to induce cytokine production, and a direct relationship exists between the number of intracellular bacteria and the amount of cytokine release [5,12]. In our experiment, we did not observe any difference in the number of internalized bacterial cells and HUVEC viability between the virulent and avirulent strains. Similar findings have been reported for other bacterial species. For example, in the case of Yersinia enterocolitica, IL-8 release was down-regulated in virulent strain-infected HUVEC but not in avirulent strain-infected HUVEC. The Yop effector protein was responsible for this inhibition of IL-8 release from HUVEC [13]. We found that S. aureus reduced IL-8 production by secreting inhibitory factor(s). IL-8 mRNA production was reduced in cytochalasin D-treated HUVEC infected with the virulent strain; furthermore, bacterial culture supernatant inhibited IL-8 production at both the protein and mRNA levels. The factor responsible for the inhibitory effect is probably a heat-stable protein, because the addition of trypsin as well as proteinase K abolished the effect of the supernatant on IL-8 production. When supernatants fractionated through Amicon filters of different molecular sizes (< 10 kDa, > 30 kDa and > 100 kDa) were added to HUVEC, IL-8 production was inhibited by the > 30 kDa fraction. However, the < 10 kDa or > 100 kDa fraction had no inhibitory effect, suggesting that the molecular mass of the inhibitory factor was 30–100 kDa (data not shown). S. aureus produces several exotoxins and modulates host immune responses. It is possible that the inhibitory factor(s) is (are) exotoxin(s). However, to the best of our knowledge, no data are available on the staphylococcal product that down-regulates IL-8 production.

These results suggest that S. aureus strains secrete certain factor(s) that inhibit IL-8 expression, and this may be related to their virulence. Because the secretion of the inhibitory factor(s) by S. aureus may result in limited bacterial elimination and prolonged bacterial survival, they may contribute to bacterial virulence. The 1-h culture supernatants of the avirulent strains (00–51 and 00–62) did not affect IL-8 production. However, supernatants obtained from the overnight culture of the avirulent strains increased IL-8 production (data not shown). IL-8 may be induced by certain bacterial factor(s) that is (are) produced during the stationary growth phase.

The present data also demonstrated that the bacterial culture supernatant of strain A17 inhibited both the basal and TNF-α-induced IL-8 expression but did not inhibit the expression of the other chemokine MCP-1. Pretreatment with bacterial culture supernatant increased TNF-α-induced MCP-1 expression. This increase may be mediated by an indirect effect or a different factor present in the supernatant; however, this remains to be clarified. This indicates that bacterial factor(s) can modulate differentially the level of some transcripts and does not inhibit gene expression globally.

Several infectious pathogens have been reported to reduce IL-8 expression. Porphyromonas gingivalis produces a proteinase that degrades chemokines; hence, P. gingivalis infection abolished IL-8 and MCP-1 production in HUVEC [14]. The Yop effector protein from Y. enterocolitica caused down-regulation of IL-8 secretion in endothelial cells [13]. Lethal toxin (LT) from Bacillus anthracis reduced IL-8 production in human endothelial cells at the mRNA level. The reduction in IL-8 production by LT was not due to the decreased transcriptional activity at the IL-8 gene promoter but to the destabilization of IL-8 mRNA [15]. Thus, inhibition of IL-8 expression resulted from either decreased transcription of the gene or mRNA instability. The activation of transcription factor nuclear factor (NF)-κB plays a key role in the production of chemotactic cytokines, including IL-8, and in cell adhesion molecule expression [16]. Several studies have demonstrated that IL-8 production was reduced by inhibition of NF-κB activation [17–19]. In our experiment, the reduction in IL-8 production in HUVEC might have been due to the down-regulation of IL-8 gene transcription or mRNA instability. However, further investigations will be required to clarify this mechanism.

In conclusion, our results indicate that the virulent S. aureus strains suppress the chemokine IL-8 expression but not MCP-1 expression. The inhibitory factor(s), i.e. heat-stable protein(s), is (are) present in the bacterial culture supernatant. Reduced IL-8 production could interfere with the host response to infection, in particular, with regard to neutrophil recruitment; hence, the production of this inhibitory factor may reflect bacterial virulence.