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Clin Exp Immunol. May 2004; 136(2): 312–319.
PMCID: PMC1809013

Differential expression of Toll-like receptor (TLR)-2 and TLR-4 on monocytes in human sepsis

Abstract

Toll-like receptors (TLRs) are a recently described family of immune receptors involved in the recognition of pathogen-associated molecular patterns (PAMPs). The central role of TLR-2 and TLR-4 in microbial responses suggests they may be implicated in the pathogenesis of human sepsis. We hypothesized that the incidence and outcome of sepsis would be influenced by the expression of TLR-2 and TLR-4 on monocytes. We have examined the expression of TLR-2 and TLR-4 mRNA and protein and their response to pro- and anti-inflammatory agents on monocytes from subjects in the intensive therapy unit (ITU) with and without Gram-negative, Gram-positive or polymicrobial sepsis. We compared these data to ITU and healthy control subjects. TLR-2 mRNA was significantly up-regulated on monocytes from subjects with both Gram-positive and Gram-negative sepsis. Similarly, we detected increased levels of TLR-2 protein on the surface of monocytes from sepsis subjects relative to ITU controls. TLR-4 mRNA was increased in Gram-positive subjects; however, there was no corresponding increase in TLR-4 protein. Although TLR-4 mRNA expression in healthy control monocytes could be modulated in vitro by culture with lipopolysaccharide or interleukin-10, this was not observed in monocytes obtained from sepsis and ITU control subjects, suggesting that septic and ITU control milieus may alter the immunoregulation of TLR-4 mRNA expression on monocytes. TLR-2 mRNA was not modulated in culture by any stimulus in any group. We suggest that expression and regulatory response of monocyte TLR-2, and to a lesser extent TLR-4 may be abnormal in human sepsis.

Keywords: endotoxin, human, inflammation, monocytes, sepsis

INTRODUCTION

Our understanding of the molecular basis of cellular responses to bacterial components has been significantly enhanced by the identification of the Toll family of proteins, of which there are at least 10 identified mammalian homologues [1]. These type I transmembrane proteins share significant similarities with the interleukin (IL)-1 receptor (IL-1R) and all contain the highly homologous toll/IL-1R (TIR) cytoplasmic domain [2]. The homology present in the TIR contrasts with significant variation in the leucine-rich repeats of the extracellular domains, which permits discrimination of a variety of pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), bacterial lipoprotein, peptidoglycans and lipoteichoic acids [1]. Of the 10 Toll-like receptors (TLRs) identified so far, TLR-2 and TLR-4 have been the most extensively studied. TLR-4 is now confirmed as the major recognition receptor for LPS [3], a component of Gram-negative organisms, whereas TLR-2 responds to bacterial lipoproteins from Gram-positive organisms [4], as well as yeasts [5] and mycobacteria [6]. Human sepsis and its most extreme form, septic shock, are commonly perceived to be caused by Gram-negative infection, but in the most recent comprehensive review of sepsis cases in the United States 52% of cases actually resulted from Gram-positive organisms, 38% of cases derived from Gram-negative infection and 5% were polymicrobial [7]. Despite recent advances in the understanding of the molecular basis of sepsis, it continues to present a major problem and the mortality rate remains high at 20–50%[7,8].

The central role of TLR-2 and TLR-4 in microbial responses suggests that they may be implicated in the incidence and outcome of human sepsis, of both Gram-positive and Gram-negative origin. Although TLR-4 is a major recognition receptor for LPS, responsiveness of TLR-2 to LPS has also been described [9]. We hypothesized that the expression of TLR-2 and TLR-4 may be altered in human subjects with Gram-positive or Gram-negative sepsis and this may be related to modification by inflammatory mediators present in sepsis. In order to explore this hypothesis we assessed first the expression of TLR-2 and TLR-4 at both the protein and mRNA level in subjects with Gram-negative, Gram-positive or polymicrobial infection requiring intensive care (as the most extreme end of the spectrum). We compared these data to both healthy subjects and intensive therapy unit (ITU) subjects without sepsis, to control for the activation of the inflammatory response present in most patients requiring intensive care. We further explored the regulatory potential of LPS, IL-10 and interferon (IFN)-γ on TLR-2 and TLR-4 monocyte expression. In summary, we detected increased levels of TLR-2 mRNA and protein expression on monocytes from sepsis subjects relative to ITU controls. However, TLR-4 mRNA was increased only in Gram-positive subjects, and there was no increase in TLR-4 protein. Although TLR-4 mRNA expression in healthy control monocytes could be modulated in vitro by culture with LPS or IL-10, this was not observed in monocytes obtained from septic and ITU control subjects. Indeed, mean TLR-4 mRNA levels were actually lower in ITU controls than healthy controls, which suggest that septic and ITU control milieus may alter the immunoregulation of TLR-4 mRNA expression on monocytes. TLR-2 mRNA was not modulated in culture by any stimulus in any group. We suggest that expression and regulatory response of monocyte TLR-2 and to a lesser extent TLR-4 may be abnormal in human sepsis.

MATERIALS AND METHODS

Subjects

Forty patients on ITU were recruited consecutively for the monocyte study. Patients with sepsis (n = 28) were defined according to the ACCP/SSCM consensus [10] and were subdivided into Gram-positive (n = 12), Gram-negative (n = 12) or polymicrobial (n = 4) according to bacterial growth in specimens. Twelve age-matched healthy subjects and 12 ITU control subjects without infection were also recruited as healthy controls and ITU controls, respectively (see Table 1).

Table 1
Patients’ characteristics at ITU admission

Monocyte purification for RNA studies

Forty ml of blood was obtained by venepuncture and collected into sterile heparinized tubes. Whole blood was diluted to twice the original volume with RPMI medium and layered onto Ficoll-Hipaque density gradient (Amersham Biosciences, Little Chalfont, UK). The gradient was centrifuged at 400 g for 30 min without braking. The resultant buffy layer was removed, RPMI medium added and then centrifuged at 200 g for 10 min. The supernatant was discarded and the pellet resuspended at 1 × 106 monocytes/ml in RPMI/10% fetal bovine serum (FBS), defined on the basis of morphology by cytospin. After 1 h adherence at 37°C, 5% CO2, the non-adherent cells were rinsed off with RPMI and the residual adherent cells (>95% monocytes defined on the basis of morphology by cytospin) were incubated for a further 2 h in medium alone or in the presence of LPS (1 µg/ml), polymyxin B (5 µg/ml), IL-10 (10 ng/ml, Peprotech, London, UK) or IFN-γ (10 ng/ml, Peprotech).

Rna extraction

Total cellular RNA was extracted from 1 × 106 monocytes after culture with stimuli as stated above. Cells were washed in sterile phosphate buffered saline (PBS) and cellular RNA extracted using RNAbee (AMS Biotechnology, Abingdon, UK) according to the manufacturer's instructions. Cellular RNA concentration was measured using a GeneQuant II (Amersham Biosciences).

Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)

RT-PCR was performed in a 20 µl one-step reaction using Reverse-IT RTase blend (ABgene, Epsom, UK) with 200 ng of total RNA as a template. RT was performed at 47°C for 30 min followed by 94°C for 2 min to inactivate the reverse transcriptase enzyme. For TLR-2 and TLR-4 amplification, PCR was performed with 30 cycles of 94°C for 20 s, 54°C for 45 s and 72°C for 1 min. For the GAPDH housekeeping gene, PCR was performed with 20 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 45 s, which ensured that the GAPDH product fell within the linear range (for specific primer sequences see Table 2). Products were electrophoresed through a 1·5% agarose gel and visualized using ethidium bromide staining. mRNA quantity was determined by digital imaging densitometry (Geldoc 1000, Bio-Rad, Hemel Hempstead, UK) (see Fig. 1).

Fig. 1
Representative RT-PCR gel. Each lane represents GAPDH, TLR-2 and TLR-4 expression in the same RNA sample. Lane 1 = ladder; lanes 2–4 Gram-positive sepsis; lanes 5–8 Gram-negative sepsis.
Table 2
Primer sequences for RT-PCR

Flow cytometry

Staining for flow cytometry was performed on peripheral blood mononuclear cells (PBMC) purified from whole blood as described above. PBMC (1 × 105) were incubated on ice for 2 min with 10 µg of human IgG, before a 30-min incubation with 5 µl of anti-CD14 FITC antibody or monoclonal anti-TLR-2 antibody, anti-TLR-4 antibody (clone TL2·1 and HTA125, respectively, Cambridge Bioscience, Cambridge, UK), or mouse IgG1 control (Dako, Ely, UK). The primary antibodies were washed off in PBS/0·5% BSA, 0·1% azide and the cells incubated for a further 30 min on ice with 2·5 µl of rabbit antimouse IgG1-pe (Dako). The secondary antibody was washed off in PBS/0·5% BSA, 0·1% azide and labelled cells were acquired on an EpicsXL flow cytometer (Beckman Coulter, High Wycombe, UK). Monocytes were gated on the basis of forward and side scatter and were >95% positive for CD14. Expression of PE-labelled TLR-2 and TLR-4 on the gated population (1 × 104 cells) was analysed using Expo 32 software (Beckman-Coulter) (see Fig. 2).

Fig. 2
Representative flow cytometry histograms depicting the monocyte expression of TLR-2 and TLR-4 (shaded) overlaid on the isotype control (white).

Statistical analysis

Semi-quantitative mRNA data are expressed as a percentage of GAPDH expression. Flow cytometry data are expressed as a ratio of isotype control fluorescence. The mRNA and flow cytometry data were normally distributed as determined by the Ryan Joiner normality test. Multiple group analyses were performed using one-way anova with Tukey's multiple comparison test or Kruskall–Wallis test with Dunn's multiple comparison test. Relationship between variables was assessed using Pearson's correlation. A P-value of < 0·05 was regarded as significant.

Results of mRNA transcripts for TLR-2 are increased in Gram-positive and Gram-negative sepsis

There was a significant difference in TLR-2 mRNA expression in monocytes between the groups (P < 0·005) (Fig. 3). The highest level of TLR-2 mRNA was detected in monocytes from subjects with Gram-positive sepsis (n = 12) (162·7 ± 30·8) and this value was significantly higher than ITU controls (82·6 ± 7·9, P < 0·05) and healthy controls (61·8 ± 1·3, P < 0·05). There was also increased TLR-2 mRNA expression in Gram-negative subjects (mean value 121·2 ± 10·2) relative to ITU controls (P < 0·05) and healthy controls (P < 0·05).There were no significant differences observed in polymicrobial sepsis compared to any of the other groups (91·5 ± 11·2).

Fig. 3
TLR-2 mRNA expression in monocytes from sepsis and control subjects. The median values are depicted by the horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively. Kruskall–Wallis test P < ...

mRNA transcripts for TLR-4 are increased in Gram-positive sepsis

There was a significant difference in TLR-4 mRNA expression in monocytes between the groups (P < 0·005) (Fig. 4). The highest levels of TLR-4 mRNA were again detected in monocytes from subjects with Gram-positive sepsis (n = 12) (219·5 ± 51·3) and this value was significantly higher than polymicrobial sepsis (90·8 ± 17·1, P < 0·05), ITU controls (71·3 ± 21·1, P < 0·01) and healthy controls (81·5 ± 1·1, P < 0·05). However, there was no increased in TLR-4 mRNA expression in Gram-negative subjects (mean value 93·8 ± 15·6) relative to any other subject group. Similarly, there were no significant differences observed in polymicrobial sepsis compared to the control groups.

Fig. 4
TLR-4 mRNA expression in monocytes from sepsis and control subjects. The median values are depicted by the horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively. One-way anovaP < 0·005. ...

TLR-2 and TLR-4 mRNA levels in monocytes are not related to survival from sepsis

Gram status had no bearing on survival in our sepsis group. Overall, our subjects with sepsis had a mortality rate of 42·8%. There were no differences in TLR-2 or TLR-4 mRNA expression relative to survival in any of the groups studied (Fig. 5).

Fig. 5
Expression of TLR-2 and TLR-4 mRNA in survivors compared to non-survivors of sepsis. The median values are depicted by the horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively.

Effect of inflammatory mediators on TLR-2 and TLR-4 mRNA expression in sepsis and non-sepsis subjects

Monocytes were incubated for 2 h alone, or with added polymyxin B, LPS, IL-10 or IFN-γ. There were no significant effects of any of the mediators on TLR-2 mRNA basal expression on monocytes from either healthy controls (n = 12), ITU controls (n = 12) or combined sepsis subjects (n = 28) (Fig. 6), although the mean level of TLR-2 mRNA expression was apparently higher in sepsis subjects than either of the control groups. By contrast, TLR-4 mRNA expression in healthy control monocytes was significantly increased by culture with LPS (P < 0·001) and significantly decreased by IL-10 (P < 0·001) (Fig. 7). Interestingly, culture of monocytes from any ITU subjects, including ITU controls had no effect on the basal level of TLR-4 mRNA expression. The mean level of TLR-4 expression was higher in sepsis subjects than either of the control groups in all culture conditions, although this did not reach statistical significance.

Fig. 6
Effect of LPS, polymyxin B (PB), IL-10 and IFN-γ on TLR-2 mRNA expression in monocytes cultured in vitro for 2 h. The median values are depicted by the horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, ...
Fig. 7
Effect of LPS, polymyxin B (PB), IL-10 and IFN-γ on TLR-4 mRNA monocytes cultured in vitro for 2 h. The median values are depicted by the horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively. ...

TLR-4 and TLR-2 mRNA levels do not correlate with LPS-induced tumour necrosis factor (TNF)-α mRNA levels

In order to determine whether the increased transcript levels observed had a functional significance we measured TNF-α mRNA, which is known to be induced by ligation of either TLR-2 or TLR-4. However, we detected no significant association between TLR-2, TLR-4 mRNA or LPS-induced TNF-α mRNA in any of the groups (data not shown).

TLR-2 expression is increased on the surface of purified monocytes from sepsis patients

Expression of surface TLR-2 and TLR-4 protein was determined by flow cytometry and the results expressed a ratio of the median fluorescent intensity obtained for the isotype control antibody. There was a significant difference in TLR-2 protein expression between the groups (P = 0·005) (Fig. 8). We detected a low level of TLR-2 expression on ITU control monocytes (2·9 ± 1·2, n = 12), which was significantly lower than expression on monocytes from Gram-negative (11·0 ± 2·8, P < 0·05) and Gram-positive subjects (13·9 ± 3·2, P < 0·05). TLR-2 protein expression was higher on monocytes from sepsis subjects than healthy control subjects (4·3 ± 2·9), although this did not quite reach statistical significance (P = 0·058). In contrast with TLR-2 expression, there were no statistically significant differences in TLR-4 protein expression between the groups studied (Fig. 9).

Fig. 8
TLR-2 protein expression on monocytes from sepsis and control subjects detected by flow cytometry. The median values are depicted by horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles respectively. One way anova ...
Fig. 9
TLR-4 protein expression on monocytes from sepsis and control subjects detected by flow cytometry. The median values are depicted by horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively, detected by ...

DISCUSSION

Although other studies have looked at human leucocyte expression of TLRs, this is the first study to examine how this expression may be altered in subjects in the ITU with and without sepsis of different origin. We detected significantly elevated levels of both TLR-2 in both Gram-positive and Gram-negative patients and TLR-4 mRNA in our Gram-positive patients. We chose to examine TLR expression on purified monocytes, which entailed collection of whole blood into heparinized tubes followed by PBMC isolation on Ficoll. It is acknowledged that such a protocol could lead to activation of the monocytes, resulting in an artificially high level of TLR expression. Indeed, a previous study by Sabroe and co-workers observed that TLR-2 and TLR-4 expression on monocytes in whole blood was several-fold lower than the expression on Ficoll-purified monocytes [10]. However, we observe differences relative to non-septic ITU controls, which suggests that monocytes from subjects with sepsis are primed for a higher level of TLR expression. To assess the functional significance of the mRNA findings, we examined whether there was any correlation between TLR expression and the expression of an LPS-responsive gene, TNF-α. Our subjects were matched closely for degree of critical illness and indeed there were no statistical differences between the groups in term of constitutive TNF-α mRNA expression. We also did not observe any correlation between TLR and LPS-induced TNF-α expression in any of our subjects. Because the protein changes we detected were restricted to TLR-2 it is possible that we may have detected a correlation if we had measured lipoprotein-induced cytokine production. However, we also did not detect any relationship with mortality, which suggests that any association between TLR expression and sepsis may be complex.

The increased level of TLR-2 mRNA is confirmed by the flow cytometry data, where we found significantly increased expression of TLR-2 protein in the Gram-negative and Gram-positive sepsis groups compared to ITU controls. Our findings are also supported by a recent study of experimental endotoxaemia in humans, where there was a sustained increase in TLR-2 protein expression on monocytes after LPS infusion [11]. This was not observed for TLR-4, where increase mRNA was detected in the Gram-positive population, but not reflected in the protein data. It has been shown previously that TLR-2 protein expression on human monocytes is regulated transcriptionally and that mRNA levels correlate with surface expression [12] and this is supported by our findings. Another study has compared mRNA and protein levels of TLR-4 and has shown that LPS can induce mRNA but actually decreases protein expression [13], although in a mouse study experimental endotoxaemia resulted in significant increases in TLR-4 protein expression but not mRNA [14]. This highlights the possibility, at least in the human scenario, that the majority of this transcript is not translated into protein. Another explanation is that ligation of receptor with LPS in the Gram-negative and polymicrobial subjects leads down-regulation of the surface expression of TLR-4, described previously as a mechanism of LPS tolerance in murine macrophages [15]. This may also be occurring in the Gram-positive subjects, as sepsis is known to increase intestinal permeability, increasing the likelihood of LPS from the gut entering the circulation [16]. Also, a recent study by Hornef and co-workers described in intestinal epithelial cells that LPS was internalized and co-localized with TLR-4, which was restricted solely to the cytoplasm in these cells [17]. LPS internalization is known to occur in human monocytes within hours [18] and it raises the possibility that may TLR-4 may be located predominantly intracellularly in Gram-positive subjects.

We did not follow-up our subjects following their ITU stay, so were unable to determine whether the alterations in TLR expression which we observed were transient or represented a predisposition to sepsis. We have, however, looked at functional polymorphisms for TLR-2 and TLR-4 in these populations and found no statistical trend (unpublished findings), suggesting that any predisposition is less likely.

We were interested to know what regulatory effect inflammatory mediators associated with the septic milieu may have on the expression of TLR-2 and TLR-4. With LPS, we incubated the LPS inhibitor polymyxin B, IFN-γ and the anti-inflammatory cytokine IL-10. One observation was that in our ITU subjects or healthy controls TLR-2 was not inducible, or inhibited by any mediators. The TLR-2 data are supported by a number of studies which demonstrate that the human form is not inducible by LPS of TNF-α, although the murine tlr2 is LPS responsive [19]. One explanation for this difference is that murine tlr2 promoter has only 10% homology with the human promoter, compared with 70% homology between the human and mouse TLR-4 promoter. Murine tlr2 contains binding sites for the transcription factors ets and SP-1, which are known to be inducible by LPS [20]. We detected the highest TLR-2 mRNA levels in the Gram-positive population where LPS presence, although unlikely to be absent, is minimal, suggesting that some other factor or a combination of factors in the sepsis milieu may be responsible for our observation. In contrast to TLR-2, we have shown that TLR-4 expression in monocytes from healthy controls can be up-regulated by LPS and down-regulated by IL-10, and this is confirmed by a number of previous studies [21,22]. A previous study has shown that IFN-γ can significantly up-regulate TLR-4 surface expression [23], but this could again be explained by the discrepancies observed between TLR-4 protein and mRNA regulation. Our ITU control subjects appear unresponsive to external stimuli and indeed the mean level of TLR-4 mRNA was slightly lower than either the sepsis subjects or the healthy controls. We know from our own unpublished observations and other studies that IL-10 is present in the plasma of ITU subjects and is an indication of the systemic inflammatory response syndrome [24]. This response may arise from a low-grade nosocomial infection for which these patients had a significant risk, due to the use of mechanical ventilation, access lines and catheters. Additionally, drugs and surgical procedures such as tracheostomy may contribute to the increase in acute inflammatory cytokines, which have been observed previously in these ‘control’ subjects [25]. It has been identified by Marchant and co-workers that high plasma IL-10 levels are correlated with sepsis severity [26], and also that IL-10 in plasma is a major monocyte deactivator in sepsis which may be detrimental to host defence [27]. This raises the possibility that IL-10 in the plasma of our ITU control subjects is sufficient to suppress TLR-4 mRNA levels, but this safety mechanism is overridden by an imbalance with other proinflammatory mediators in septic plasma. The question remains as to whether increased IL-10 is an example of dysregulation or normal homeostatic response to control inflammation, as we have described previously [28].

In summary, we have found increased levels of TLR-2 mRNA and protein and increased levels of TLR-4 mRNA in subjects with sepsis compared to healthy and ITU controls. These increases were most marked in the Gram-positive population, suggesting that LPS tolerance may compromise the expression of Toll-like receptors in Gram-negative sepsis. TLR-4 mRNA levels were resistant to in vitro immunomodulation, constitutively high in sepsis subjects and constitutively low in ITU control subjects. This implies that the sepsis and non-sepsis milieus may lead to up-regulation and suppression of TLR-4 mRNA, respectively. The lack of association between TLR-2 and TLR-4 expression on monocytes with any functional outcome suggests, however, that any relationship between TLR expression and human sepsis may be complex.

Acknowledgments

The authors wish to acknowledge the assistance of staff of ITU at Southmead Hospital, North Bristol NHS Trust and Dr Mark Perry, University of Bristol for statistical advice. Dr Lynne Armstrong is supported by Action Research.

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