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. 2004 Feb;135(2):336–342. doi: 10.1111/j.1365-2249.2004.02351.x

Mycobacterial heat shock protein 70 induces interleukin-10 production: immunomodulation of synovial cell cytokine profile and dendritic cell maturation

T DETANICO *, L RODRIGUES †, A C SABRITTO †, M KEISERMANN †, M E BAUER †, H ZWICKEY ‡, C BONORINO †
PMCID: PMC1808950  PMID: 14738465

Abstract

Cytokines are key modulators of the immune responses that take place in the inflamed synovium of arthritis patients. Consequently, substances that can reverse the inflammatory profile of the inflamed joint are potential tools for clinical management of the disease. Mycobacterial heat shock protein 70 (MTBHSP70) has been found to protect rats from experimentally induced arthritis through the induction of interleukin (IL)-10-producing T cells. In this study, we have demonstrated that MTBHSP70 induces IL-10 production in synoviocytes from arthritis patients and peripheral blood monoculear cells (PBMCs) from both patients and healthy controls. IL-10 production was accompanied by a decrease in tumour necrosis factor (TNF)-α production by synovial cells. Separation studies showed that the target cells were mainly monocytes. Accordingly, we observed that MTBHSP70 delayed maturation of murine bone marrow-derived dendritic cells. Our results suggest that MTBHSP may act on antigen-presenting cells (APCs) to modulate the cytokine response in arthritis and support an anti-inflammatory role for this protein, suggesting that it may be of therapeutic use in the modulation of arthritis.

Keywords: arthritis, cytokines, dendritic cells, HSP70, Mycobacterium tuberculosis

INTRODUCTION

Cytokines modulate the entire course of immune responses that take place in the inflamed synovium. In particular, inflammatory cytokines contribute to the persistence of arthritis, and arthritis can be ameliorated by neutralization of these molecules. For instance, tumour necrosis factor (TNF)-α is the major inflammatory cytokine produced by macrophage-like synovial cells and its neutralization by anti-TNF-α antibodies are a current therapeutic strategy for the disease [1]. Interferon (IFN)-γ is the most abundant cytokine produced by synovial T cells of arthritis patients, and administration of IFN-γ can exacerbate autoimmunity [2]. Interleukin (IL)-10 is known to down-regulate the effects of these two other cytokines [3]. The production of IL-10 is correlated negatively with arthritis progression and joint destruction [4]. Also, children with arthritis have been reported to have a hereditary predisposition to low IL-10 production [5]. Consequently, IL-10 is recognized as a major anti-inflammatory molecule, and is considered for therapeutic purposes in arthritis and other diseases.

Substances that can influence immune responses and are able to reverse the inflammatory profile of the synovium are also candidate therapeutic agents for arthritis. Mycobacterial heat shock protein 70 (MTBHSP70) has a number of immunological properties. It has been shown to be an excellent adjuvant for antibody production [6], as well as an extremely powerful antigen, even in lipopolysaccharide (LPS) hyporesponsive mice [7]. Finally, immunization with MTBHSP70 has been shown to protect against adjuvant arthritis in rats [8]. It was demonstrated later that arthritis protection was due mainly to the induction of IL-10-producing T cells that recognize a specific MTBHSP70 peptide [9,10].

In this study, we show that LPS-free MTBHSP70 can induce IL-10 production in blood and synovial cells of arthritis patients. This is accompanied by down-regulation of IFN-γ and TNF-α production by the same cells. The responder cells are shown to be mainly monocytes. Finally, we demonstrate that MTBHSP70 can also delay maturation in murine bone marrow derived dendritic cells, suggesting an anti-inflammatory potential for this protein.

MATERIALS AND METHODS

Subjects

Written informed consent was obtained from all subjects after receiving ethical approval from the University Hospital (PUCRS, Porto Alegre, Brazil). Blood and synovial fluid was obtained from 14 out-patients (mean age = 50, range 43–62) diagnosed by an experienced rheumatologist at the rheumatology unit. Five patients were diagnosed with rheumatoid arthritis and nine with reactive arthritis. All subjects were chronically ill out-patients recruited from the rheumatology service, and were experiencing a relapse with acute inflammation when synovial fluid was harvested. The patients were under different medication regimens (Table 1). Responses were compared only among patients diagnosed with the same disease. For some experiments, blood was also obtained from healthy control volunteers who worked in the laboratory (mean age = 28, range 20–35)

Table 1.

Characteristics of the study sample; a total of 14 patients was analysed in this study and treated according to diagnosis (M = male, F = female)

Diagnosis Gender Mean age (years) Treatment
Reactive arthritis 6M, 3F 49 Doxicycline 100 mg 2× day andprednisone 5 mg 2× day
Rheumatoid arthritis 5M, 2F 51 Prednisone 12·5 mg/day or 5 mg 2× dayMetothrexate 7·5 mg/week
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Peripheral blood mononuclear cell isolation

Twenty ml of peripheral blood was collected by venepuncture in the morning (between 8 and 10 a.m) and samples stored into heparinized tubes. Synovial fluid (approximately 10 ml) was collected from all patients and diluted immediately 4 : 3 with phosphate buffered saline (PBS) and 15 mm EDTA before analysis. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over a Ficoll-Hypaque (Sigma, St Louis, MO, USA) gradient (900 g for 30 min). Mononuclear synovial cells were similarly isolated. Cell viability was assessed by trypan blue (Sigma) dye exclusion and was always greater than 95%. Cell purification was performed either by adherence to plastic or using the magnetic bead system (Miltenyí Biotec, Auburn, CA, USA). For adherence separation, synovial cells were cultured for 1 h in 24-well plastic plates (TPP, Switzerland), and then the non-adherent cells were washed out gently and transferred into another well. Blood monocytes and T cells were purified using magnetic beads for negative selection (pan T cell isolation kit, 130-053-001; monocyte isolation kit, 130-053-301) and 95–98% of purity was obtained, as determined by FACS analysis.

Reagents and LPS contamination control

Dexamethasone (D4902) and LPS (L-2630) were purchased from Sigma. Dexamethasone was reconstituted in ethanol and used at 10–5−10–9m. Bovine serum albumin was purchased from Invitrogen (Carlsbad, CA, USA; 11018–017). Recombinant MTBHSP70 was purified from plasmid PY3111 (a gift from Dr Douglas Young, Hammersmith Hospital, London, UK) in our laboratory according to Mehlert [11]. Because a major limitation of studies on cytokine stimulation by HSPs has been potential contamination with LPS [12,13], purified protein was always screened carefully for LPS and the preparations used to stimulate the cell cultures were LPS-free. Contaminant LPS was removed using Triton X-114, according to the method described in Aida and Pabst [14]. LPS levels were always monitored using the limulus amoebocyte (LAL) assay (50–648 U, Biowhittaker, Walkersville, MD, USA). MTBHSP70 was used only when LPS levels were below 0·005 EU/ml. Integrity of MTBHSP70 after Triton extraction was assessed by monitoring its ATPase activity in the presence of Mg++, using a method described in [15].

Human cell cultures

The synovial and PBMCs were cultured in a final concentration of 8 × 105 cells/ml in complete culture medium (RPMI-1640 media supplemented with gentamicin 0·5%, glutamine 1%, hepes 1% and fetal calf serum 10%, all from Sigma) for 48 h at 37°C in 5% CO2 atmosphere. Cells were stimulated with either 1% phytohaemagglutinin (PHA) (Gibco BRL, Grand Island, NY, USA) or 1 µg/ml recombinant Mycobacterium tuberculosis HSP70 or bovine serum albumin (BSA).

Murine dendritic cell cultures

C57Bl/6 mice were purchased from LACEN (Rio Grande do Sul, Brazil). Dendritic cells were grown from bone marrow with GM-CSF and IL-4, as described by Inaba et al. [16] and used on day 5 of culture, still immature as assessed by FACS analysis of class II and B7 expression. The cells were incubated with either dexamethasone, LPS, MTBHSP70 or BSA for 48 h and then analysed for maturation by FACS. The supernatant was collected and used for cytokine analysis.

Cytokine analysis and FACS

Commercially available enzyme-linked immunosorbent assay (ELISA) kits (Quantikine or Duo-Set, R&D Systems, Minneapolis, MN, USA) were used to measure human or murine cytokine concentrations in cell culture supernatant. The following cytokines were measured: IFN-γ, IL-10 and TNF-α. The λ450 was detected using an ELISA plate reader (Biorad, Hercules, CA, USA) and concentrations extrapolated from a log-transformed curve (GraphPad Prism 3·0, San Diego, CA, USA). Data are expressed in pg/ml. Antibodies for FACS were purchased from Pharmingen.

RESULTS

Effect of HSP70 stimulation on cytokine production

Table 1 lists the demographic and clinical information from patients enrolled in the study. A total of nine males and five females were diagnosed with either rheumatoid arthritis (RA) or reactive arthritis (ReA). The RA and ReA patients were being treated with corticosteroids; RA patients were being treated with metotrexate and ReA patients with doxicycline.

Previous studies have demonstrated that T cells are less able to produce IL-10 in arthritic patients [17,18]. We therefore tested cytokine production of PBMCs in PHA-stimulated cultures, in which T cells are expanded polyclonally. PHA-stimulated cultures produce IFN-γ in both healthy controls and patients of RA or ReA, as shown in Fig. 1a. While all three groups produce IL-10 in response to PHA, arthritis patients produce much less, suggesting an inherent difference in the cells from arthritis patients. There were no detectable amounts of these cytokines in unstimulated cultures (data not shown).

Fig. 1.

Fig. 1

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Effect of MTBHSP70 on PBMC cytokine production. PBMCs from rheumatoid arthritis (RA), reactive arthritis (ReA) patients or healthy controls were isolated and cultured 8 × 10 5/ml for 48 h with either phytohaemagglutinin (PHA) 1% (a) or mycobacterial HSP70 (MTBHSP70) 1 µg/ml (b). Cytokines were assayed in the supernatant by ELISAs. Lines represent mean.

Because HSP70 has been shown to induce IL-10 production in animal studies, we next investigated whether MTBHSP70 treatment in vitro could regulate cytokine production in blood cells from arthritis patients. As shown in Fig. 1b, MTBHSP70 treatment induced significant IL-10 production in PBMCs from RA and ReA patients as well as from normal controls. That MTBHSP70 stimulates IL-10 production and PHA does not suggest that the IL-10 is not being made by T cells in the culture. Interestingly, incubation with MTBHSP70 led to significant production of IL-10, but not IFN-γ. The up-regulation of IL-10 was more pronounced in PBMCs from arthritis patients than in those from control subjects.

To determine if the microenvironment of the joint, like PBMCs, was sensitive to MTBHSP70, we incubated synovial cells from five reactive arthritis patients with BSA, LPS or MTBHSP70 (Fig. 2). Cells cultured with BSA (used here as an innocuous control) showed TNF-α as the dominant cytokine. Although TNF-α could already be observed in 24 h cultures with BSA, it was not observed in the wells treated with MTBHSP70 (data not shown). Incubation with MTBHSP70 led to a reversal of this inflammatory profile − an induction of IL-10 correlated with a decrease in TNF-α and IFN-γ production. Although incubation with LPS led to a decrease in TNF-α production, it did not induce an increase in IL-10. MTBHSP70 up-regulation of IL-10 production was even greater in synovial cells compared to what was observed in PBMCs from patients. Cell death was not observed in any of the cultures until 72 h, when a massive decrease in size and increase in granularity could be observed by FACS analysis both in the LPS and MTBHSP70 cultures (data not shown).

Fig. 2.

Fig. 2

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Cytokine production by synovial cells upon stimulation with MTBHSP70. Synovial cells of six reactive arthritis patients were cultured 8 × 105/ml for 48 h with either 1 µg/ml MTBHSP70, 1 µg/ml BSA or 60 EU of LPS. Data expressed as mean ± s.e.

Monocytes are the major source of MTBHSP70-induced IL-10

In order to identify the target cells that were being stimulated to produce IL-10 by MTBHSP70, we performed cell separation studies. Figure 3 shows the representative results of three of these experiments. Synovial cells from one male (Fig. 3a) and one female (Fig. 3b) reactive arthritis patients were separated by adherence to plastic and incubated with PHA, MTBHSP70 or BSA, and IL-10 was measured in the supernatant. Non-adherent cells produced high levels of IFN-γ in response to PHA, but not to MTBHSP70 (data not shown). However, while non-adherent cells were the main producers of IL-10 in response to PHA, adherent cells were the major producers of IL-10 in response to MTBHSP70. A similar phenomenon was observed in PBMCs of a male healthy control. PBMCs were separated by negative selection magnetic beads and incubated with either BSA or MTBHSP70. While an increase in IL-10 production was observed by unseparated cells in response to MTBHSP70, it was not observed in the isolated T cell population under the same stimulus (Fig. 3c). In contrast, IL-10 production was observed clearly in the isolated monocytes (Fig. 3d). Similar results were obtained with the synovial cells of one female lupus patient and monocytes of one female healthy control (data not shown).

Fig. 3.

Fig. 3

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MTBHSP70 induced IL-10 production by synovial adherent cells and blood monocytes. Synovial cells of (a) one male and (b) one female reactive arthritis patient were fractionated by adherence to plastic and cultured with PHA, MTBHSP70 or BSA. ad = adherent cells; nad, non-adherent cells. (c, d) PBMCs of one male healthy control were fractionated using negative selection magnetic beads for T cells (b) or monocytes (c) cultured for 48 h with either BSA or MTBHSP70.

Enriching for monocytes yielded similar levels of MTBHSP70-induced IL-10 in patients’ synovial cells and healthy control PBMCs (Fig. 3a,b,d), while no increases in IL-10 were observed in the T cell-enriched populations. This observation suggests that the differences observed in IL-10 up-regulation by MTBHSP70 in blood and synovium might, at least in part, be explained by the number of target cells. Monocytes/macrophages constituted 28·0 ± 0·9% of the synovial cells, as assessed by CD14 expression (n = 4), versus 7·1 ± 0·8% in patients (n = 3) and 4·3 ± 0·25% in healthy control blood (n = 4). Interestingly, simply removing the T cells and enriching for monocytes in PBMCs in control blood led to an increase in the amount of IL-10 compared to unseparated, BSA-treated cells (Fig. 3d).

MTBHSP70 induced delayed maturation of murine dendritic cells

The induction of IL-10 by HSP70 as well as the modulation of the inflammatory profile of synovial cells appeared to be conserved between different arthropathies as well as in normal blood monocytes. We then hypothesized if it would also be conserved in an animal model, modulating other phenomena that depended on inflammatory signals, such as dendritic cell maturation. To test that hypothesis, murine bone marrow-derived immature dendritic cells were incubated with BSA, MTBHSP70, LPS or dexamethasone. The results are summarized in Fig. 4. On day 7 of culture, dendritic cells incubated with LPS-free control BSA exhibited minimal spontaneous maturation, whereas incubation with LPS enhanced maturation as assessed by both CD86 and MHC class II expression (Fig. 4a). In contrast, incubation with MTBHSP70 inhibited spontaneous maturation (Fig. 4b). Similarly, the synthetic glucocorticoid dexamethasone inhibits spontaneous maturation of dendritic cells (Fig. 4c). To verify if MTBHSP70 could also inhibit LPS-induced maturation, we added both LPS and MTBHSP70 to the cell cultures. MTBHSP70 inhibits LPS-induced maturation when compared to dendritic cell cultures incubated with LPS alone (Fig. 4d).

Fig. 4.

Fig. 4

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MTBHSP70 delayed maturation of murine dendritic cells. Bone marrow-derived dendritic cells were cultured as described in the Methods section and incubated on day 5 with either 60 EU of LPS (a), 12 µg/ml MTBHSP70 (b) 10−7m dexamethasone (c). Maturation (dark line) was analysed by MHC class II (left) and CD86 (right) expression, over the baseline maturation at day 7 observed with BSA incubation. (d) Cells cultured with both 60 EU of LPS and 12 µg/ml of MTBHSP70. Inhibition of maturation is shown in the dark line, over the LPS alone-induced maturation.

We measured cytokine production by the dendritic cells to determine if IL-10 alone was responsible for delayed dendritic cell maturation. MTBHSP70 and dexamethasone alone induced IL-10, but not TNF-α in the dendritic cells (Fig. 5). LPS alone caused significant TNF-α production as well as induction of IL-10 in similar levels to MTBHSP70 and dexamethasone. In the cultures that were stimulated with both LPS and MTBHSP70, there was a decrease in TNF-α production compared to the cultures stimulated exclusively with LPS.

Fig. 5.

Fig. 5

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Cytokine production by dendritic cell cultures. Bone marrow-derived dendritic cells were cultured as described in the Methods section and incubated on day 5 with either BSA, MTBHSP70, LPS, dexamethasone (DEX) or LPS plus MTBHSP70. Cytokines were measured by ELISA 48 h later.

DISCUSSION

The striking induction of IL-10 by MTBHSP70 in synovial cells agrees with previous findings regarding experimental arthritis models in rats, in which protection could be induced by immunization with this protein [9,10,19,20], leading to the induction of IL-10-producing T cells. Other animal studies, including rat listeriosis [21] and bovine tuberculosis [22], implicated MTBHSP70-reactive, IL-10-producing CD4+ T cells in the termination of excessive inflammation during infection, suggesting that HSPs are important modulators of the inflammatory response [23].Our human study results support these previous animal studies. The priming and differentiation of T helper cells depends on the signals and cytokines provided by antigen-presenting cells. IL-10-producing APCs induce differentiation of T CD4 cells into IL-10-producing T cells [24]. In particular, the state of maturation of dendritic cells as well as the cytokines they produce are known to not only drive differentiation into TH1 or TH2 profiles [25,26], but also to ensue T cell tolerance or activation [27,28].

Our results show that MTBHSP70 can act on human antigen-presenting cells, delivering anti-inflammatory signals and leading to IL-10 production. It can thus reverse the inflammatory profile of the synovial cells, leading to a decrease in IFN-γ and TNF-α production and an increase in IL-10 production, as can be seen in Fig. 2. This phenomenon is likely to initiate in vivo the priming of T cells that will develop into an anti-inflammatory profile, such as those described in the animal models, leading to amelioration of arhtirtis. The fact that we have not observed T cells as the major target of MTBHSP70 in our in vitro assay, as well as the induction of cell death after 72 h of culture, suggests that further events are necessary in vivo to generate such regulatory cells. Finally, the bias towards an anti-inflammatory profile for MTBHSP70 is in agreement with all the previous findings on its antigenic and adjuvant abilities, which usually point to antibody responses, driven by TH2-like cytokines.

Interestingly, synovial cells did not produce TNF-α in response to challenge with LPS (Fig. 2), indicating that that cells from arthritis patients are refractory to inflammatory stimuli. A state similar to endotoxin tolerance has been described in the absence of infection, such as in cardiogenic shock [29]. Also, MTBHSP70 induction of IL-10 was more pronounced in synovial cells from patients than in their PBMCs, and PBMCs from arthritis patients produced significantly more IL-10 in response to MTBHSP70 than the PBMCs from normal controls (Fig. 1b). Although in part these differences can be explained by the number of monocytes in the cultures, these results suggest that cells from the patients may also be in a different state of activation, more sensitive to the anti-inflammatory signals provided by MTBHSP70. For example, a difference in the adhesive properties of circulating monocytes in PBMCs from psoriasis patients versus control subjects has been described [30], alongside with increased inflammatory cytokine production.

The anti-inflammatory potential of MTBHSP70 proved to be conserved in an animal model. In mice, MTBHSP70 delayed maturation of bone marrow-derived murine dendritic cells (Fig. 4), while treatment with LPS promoted maturation. That both treatments induced similar levels of IL-10 production by the cells (Fig. 5) suggests that the induction of IL-10 is not the sole anti-inflammatory signal delivered by MTBHSP70. This result is inconsistent with data reported previously by Wang et al. [31], due most probably to all the differences in the methodology (origin of cells, amount of MTBHSP70 used for stimulation, LPS removal strategy). Indeed, differences in the responses to LPS have been reported for bone marrow- versus monocyte-derived dendritic cells [32]. Opposite responses have been reported for treatment with high- versus low-dose HSP [15], and even for treatment with different peptides of MTBHSP70 [33]. Taken together, all the studies commented upon here indicate strongly that immune responses are exquisitely sensitive to modulation by MTBHSP70. We are currently studying the effect of different doses of MTBHSP70 in human monocyte-derived dendritic cells.

Finally, it is noteworthy to mention that the phenomena described here were equivalent in patients with different arthropathies, undergoing different pharmacological treatments. Antibiotics [34] and anti-inflammatory drugs, both steroidal [35,36] and non-steroidal [37], as well as other commonly used drugs are known to have multiple, sometimes undesired, effects on cytokine production. Our results suggest that MTBHSP70 has significant immunomodulatory effects, with potential uses in varied medical conditions, even in combination with other drugs.

Acknowledgments

This work was supported by grants from CNPq and FAPERGS. We would like to thank the excellent technical assistance of Ingrid Porto, Adriana Mota and Carla Schmitz.

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