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. 2007 Feb;147(2):370–378. doi: 10.1111/j.1365-2249.2006.03288.x

Mycobacterium bovis bacillus Calmette–Guérin (BCG) induces human CC- and CXC-chemokines in vitro and in vivo

Y Luo 1, X Chen 1, M A O'Donnell 1
PMCID: PMC1810474  PMID: 17223980

Abstract

Both CC- and CXC-chemokines are known to be potent leucocyte activators and chemoattractants and play important roles in inflammatory responses. However, chemokine response to bacillus Calmette–Guérin (BCG) infection remains incompletely defined. In this study, we investigated human CC- [macrophage-derived chemokine (MDC), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α and eosinophil chemoattractant activity (eotaxin)] and CXC-interferon-inducible protein (IP)-10 chemokine production in response to BCG stimulation. BCG efficiently induced all chemokines tested in the urine of four bladder cancer patients undergoing intravesical BCG immunotherapy. The peak urinary chemokine responses occurred generally between the fourth and sixth weekly treatment, except eotaxin, which was less predictable. To evaluate the effect of BCG on induction of chemokines in vitro, urothelial cell lines and peripheral blood mononuclear cells (PBMCs) were used. Although BCG induced no or marginal chemokines from urothelial SV-HUC-1, RT4 and T24 cells, BCG-derived cytokines [interleukin (IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)-α] induced all chemokines tested except eotaxin from these cell lines. BCG also efficiently induced all chemokines tested except eotaxin from PBMCs of both BCG-naive and BCG-vaccinated subjects. MCP-1 and MIP-1α emerged at 4–5 h post-BCG exposure (early chemokines); IP-10 elevated at day 1 and peaked at day 2 (intermediate chemokine); and MDC elevated at day 1 and peaked at day 7 (late chemokine). This kinetic pattern was paralleled with that of BCG-induced cytokines [early: TNF-α; intermediate: IL-6 and IL-10; and late: IFN-γ and granulocyte–macrophage colony-stimulating factor (GM-CSF)]. Taken together, these results indicate that BCG directly or indirectly induces human CC- and CXC-chemokine production, which may represent one of the mechanisms by which BCG exerts its anti-tumour activity.

Keywords: BCG, bladder cancer, chemokine, cytokine

Introduction

Chemokines are a group of polypeptides of low molecular weight that belong to the CC, CXC, C or CX3C family according to the organization of positionally conserved cysteine residues in the amino acid sequence [1]. The vast majority of the chemokines identified so far belong to the CC and CXC subfamilies. Chemokines are produced by many different cell types after stimulation with appropriate inducers, including endothelial cells, epithelial cells, fibroblasts, monocytes/macrophages, lymphocytes, neutrophils, etc. [1–3]. Chemokines play important roles in the regulation of host responses to inflammatory stimulation. In general, chemokines of the CC family mainly attract and activate monocytes/macrophages, lymphocytes, basophils, eosinophils, natural killer (NK) cells and dendritic cells, whereas CXC chemokines mainly attract and activate neutrophils, but some also activate T cells and NK cells [1–3]. Chemokines can also be produced by tumour cells [4] and be associated with certain disease states when aberrantly expressed [5].

Bacillus Calmette–Guérin (BCG) has been used for many years as the world's most widely used tuberculosis vaccine. BCG has also been used to treat superficial transitional cell carcinoma (TCC) of the bladder for more than two decades [6–8]. Although the exact mechanisms by which BCG mediates anti-bladder cancer immunity remain unclear, a local non-specific immunological reaction reflecting the activation of various cell types has been suggested [7–9]. Following intravesical BCG instillation immune cell infiltration in the bladder wall of patients has been observed, including T cells, NK cells and macrophages [10–13]. A large number of immune cells in patients' voided urine after intravesical BCG therapy has also been observed to include neutrophils, T cells and macrophages [14–16]. Moreover, a transit secretion of cytokines in patients' urine after intravesical BCG therapy has been observed, including interleukin (IL)-1, IL-2, IL-6, IL-10, IL-12, IL-18, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and granulocyte–macrophage colony-stimulating factor (GM-CSF) [16–23]. In addition, studies have also shown excreted urinary CXC chemokines after intravesical BCG therapy, including IL-8 (CXCL8) and interferon-inducible protein 10 (IP-10/CXCL10) [20–24]. Although the specific role each of these proinflammatory mediators in orchestrating BCG-induced anti-tumour immunity is unclear, a high expression of T helper type (Th) 1 cytokines (IL-2, IL-12 and IFN-γ) has been observed to be associated with BCG responders, whereas a high level of Th2 cytokine IL-10 appears to be associated with BCG failures [18,19,25,26]. Thus, the pattern of urinary cytokines may determine specific types of immune response to BCG therapy, while such a cytokine pattern may actually be affected by various chemokines produced in situ.

In this study, we investigated human CC-chemokine responses to BCG stimulation, including macrophage-derived chemokine (MDC/CCL22), monocyte chemoattractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein-1α (MIP-1α/CCL3) and eosinophil chemoattractant activity (eotaxin/CCL11). We also investigated IP-10 (CXCL10) response to BCG stimulation in this study. The selected CC- and CXC-chemokines reflect the major cellular sources and targets currently known for chemokines [1–3]. We evaluated BCG's ability to induce these chemokines from healthy human peripheral blood mononuclear cells (PBMCs), the bladder wall of patients with bladder TCC and the commonly used human urothelial cell lines in vitro and in vivo. Our results demonstrate that BCG is a potent CC- and CXC-chemokine inducer, and that BCG's anti-bladder cancer effect may depend on production of the investigated chemokines.

Materials and methods

BCG

MV261 BCG (BCG), a Pasteur strain transfected previously with the kanamycin resistance plasmid pMV261 [27], was used in the in vitro experiments. This strain has demonstrated the similar immunostimulatory property to that of commercial lyophilized BCG preparations. This BCG strain was kept routinely at 37°C in 7H9 Middlebrook broth (Difco, Detroit, MI, USA) supplemented with 10% albumin dextrose catalase [ADC: 5% bovine serum albumin (BSA), 2% dextrose and 0·85% NaCl], 0·05% Tween 80 (Sigma, St Louis, MO, USA) and 30 µg of kanamycin per ml. One unit of absorbance at 600 nm for the BCG culture was calculated as 2·5 × 107 colony-forming units (CFUs). For clinical intravesical therapy, lyophilized BCG preparations (TheraCysTM, Connaught Pasteur Merieux, Inc., Toronto, Ontario, Canada) were used.

PBMC culture

In accordance with the approved clinical protocol at our institution, blood samples were collected from both BCG-naive and BCG-vaccinated healthy donors with negative and positive skin test reactivity to the purified protein derivative (PPD), respectively. PBMCs were prepared from buffy coat leucocytes purified on Ficoll-Paque (Pharmacia, Uppsala, Sweden). Viability by trypan blue exclusion exceeded 95%. PBMCs were suspended in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and 30 µg of kanamycin per ml, and incubated at 37°C in a humidified 5% CO2 incubator at a density of 5 × 105 cells/200 µl per well in 96-well tissue culture plates in the presence or absence of BCG (0·01 OD/ml). The plates were incubated for various time-points and then frozen at −20°C until the enzyme-linked immunosorbent assay (ELISA) was performed.

Urothelial cell lines and cultures

Both human normal urothelial cell line (SV-HUC-1) and bladder cancer cell lines (RT4 and T24) were obtained from American Type Culture Collection (Rockville, MD, USA). The SV-HUC-1 line is an SV40 immortalized normal ureter epithelial cell line; the RT4 line is a well-differentiated bladder TCC cell line; and the T24 line is a poorly differentiated bladder TCC cell line. All these cell lines were cultured routinely in RPMI-1640 medium containing 10% FBS, l-glutamine (2 µM), penicillin-G (100 U/ml) and streptomycin (100 µg/ml) at 37°C with 5% CO2. For induction of chemokines, 2 × 105 cells were seeded overnight per well in 24-well plates. BCG (0·01 OD/ml), recombinant (r) IL-1β (1 ng/ml; Roche Molecular Biochemicals, Indianapolis, IN, USA), rIFN-γ (10 ng/ml; Roche Molecular Biochemicals) or rTNF-α (10 ng/ml; Endogen, Cambridge, MA, USA) were then added and the incubation continued for 3 days. Supernatants were harvested after incubation and stored at −20°C until ELISA analysis.

Urine samples

Urine samples were collected from four bladder cancer patients undergoing the 6-weekly intravesical BCG immunotherapy after transurethral resection of their bladder tumours (TURBT). These patients consented with our Institutional Review Board (IRB) approval. Previously, we assayed voided urine samples for cytokines collected from various times after intravesical BCG immunotherapy and found that over 90% of urinary cytokines emerged within the first 2–12 h after therapy [26,28,29]. Thus, voided urine during that time-period was collected and pooled for later analysis. Urine samples were stabilized during patient collection with a concentrated buffer containing 2 M Tris-HCl (pH 7·6), 5% BSA, 0·1% sodium azide and four protease inhibitors (aprotinin, pepstatin and leupeptin at 0·01 µg/ml for each and aminoethylbenzenesulphonyl fluoride (AEBSF) at 0·1 µg/ml, all purchased from Sigma). At the end of collection, the volume of the 10-h urine was recorded. A 10-ml sample was preserved further by the addition of a protease inhibitor cocktail tablet (Roche Molecular Biochemicals) and then stored at −20°C prior to batch analysis by ELISA.

ELISA analysis and reagents

ELISA reagents including recombinant human chemokines and cytokines and paired monoclonal capture and detecting antibodies were obtained from R&D Systems (Minneapolis, MN, USA) for MDC and MIP-1α, from Endogen (Cambridge, MA, USA) for IL-6, IL-8, GM-CSF and IFN-γ, and from PharMingen (San Diego, CA, USA) for MCP-1, IP-10, eotaxin, IL-10 and TNF-α. A sandwich format ELISA was performed according to the manufacturer's instructions.

Statistical analysis

All determinations were made in duplicate and each result was expressed as mean ± standard deviation (s.d.). Statistical significance was determined by paired Student's t-test. A P-value of 0·05 was considered significant.

Results

Urinary chemokine production in response to intravesical BCG therapy

To evaluate the effect of BCG on chemokine production in vivo, we analysed excreted urinary chemokines in four bladder cancer patients undergoing intravesical BCG immunotherapy. These patients received the standard 6-weekly BCG instillation 2–6 weeks after TURBT. Urine was collected before and between 2 and 12 h after each of the 6-weekly intravesical BCG instillations. ELISA was used to analyse urinary MDC, MCP-1, MIP-1α, IP-10 and eotaxin. Among the four patients tested, all patients showed no or marginal production of chemokines prior to BCG treatment (Fig. 1). However, after BCG treatment, all four patients showed increased urinary IP-10, three patients (numbers 1, 3 and 4) showed increased urinary MDC, MCP-1 and eotaxin, and two patients (numbers 1 and 4) showed increased urinary MIP-1α. The peak urinary chemokine responses except eotaxin occurred between the fourth and sixth weekly BCG treatment for patients 1, 2 and 4 and the second weekly BCG treatment for patient 3. The peak urinary eotaxin response was less predictable. Among the five chemokines tested, IP-10 was the most dominant chemokine found in the urine after intravesical BCG instillation. Patient 1 received a second course of the 6-weekly intravesical BCG treatment 3 months later due to his failing initial BCG therapy. Urine was collected for ELISA analysis of chemokines, as above (Fig. 2). Urinary IFN-γ and IL-10 were analysed in parallel for comparison, as these cytokines were known to be potential surrogate markers for clinical response to intravesical BCG immunotherapy [18,19,25,26]. This patient became a BCG responder after the second course of therapy. In accordance with his clinical improvement, he developed early and sustained urinary chemokines and cytokines during the second course of BCG therapy (Fig. 2). The ratio of his urinary IFN-γ to IL-10 was also increased. These results indicate that BCG is potent on induction of chemokines other than cytokines in vivo via intravesical application.

Fig. 1.

Fig. 1

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Bacillus Calmette–Guérin (BCG)-induced urinary chemokine production in bladder cancer patients undergoing intravesical BCG immunotherapy after transurethral resection of their bladder tumours (TURBT). The urine of bladder cancer patients (numbers 1–4) was collected before and 2–12 h after each 6-weekly BCG instillation and analysed by enzyme-linked immunosorbent assay (ELISA) for chemokine production [macrophage-derived chemokine (MDC), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), interferon-inducible protein (IP) and eosinophil chemoattractant activity (eotaxin)]. The values are expressed as the total chemokine mass per 10-h urine.

Fig. 2.

Fig. 2

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Bacillus Calmette–Guérin (BCG)-induced urinary chemokine production in a bladder cancer patient who failed the initial BCG therapy but became a BCG responder after a second BCG therapy. Patient 1 shown in Fig. 1 received a second course of the 6-weekly intravesical BCG instillation 3 months after initial BCG therapy. The patient's urine was collected and processed for enzyme-linked immunosorbent assay (ELISA) analysis of chemokines [macrophage-derived chemokine (MDC), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), interferon-inducible protein (IP) and eosinophil chemoattractant activity (eotaxin)] and cytokines [interferon (IFN)-γ and interleukin (IL)-10]. For comparison, his urinary chemokines and cytokines found during the initial BCG therapy are included. The values are expressed as the total chemokine/cytokine mass per 10-h urine. Letters I and I′ stand for initial and secondary induction therapy, respectively. The clinical status of the patient in response to BCG therapy is indicated in italics. A BCG non-responder refers to any patient who relapses within 6 months of starting a 6-week induction cycle of BCG. A BCG responder is any patient who attains at least a 6-month disease-free period after starting an induction cycle of BCG.

Chemokine production by urothelial cell lines in response to BCG and BCG-derived cytokines

To determine whether urothelial cells could contribute to urinary chemokines observed after intravesical BCG immunotherapy, we made use of three commonly used human urothelial cell lines SV-HUC-1 (a normal urothelial line), RT4 (a well-differentiated TCC line) and T24 (a poorly differentiated TCC line). Cells were stimulated in culture with a predetermined dose of BCG or selected cytokines found in the urine after intravesical BCG immunotherapy (i.e. IL-1β, IFN-γ or TNF-α) for 3 days. Chemokine production was analysed by ELISA including MDC, MCP-1, MIP-1α, IP-10 and eotaxin (Fig. 3). Both IL-6 and IL-8 (CXCL8) were included for comparison as they were known to be produced from T24 cells in culture [30–33]. Among the chemokines tested, eotaxin was undetected in all three cell lines under the experimental settings. SV-HUC-1 cells were found to secrete spontaneously MDC, MCP-1, MIP-1α and IP-10 during 3-day culture. Although BCG increased no or marginal chemokines tested in SV-HUC-1 cells, BCG-derived cytokines (IL-1β, IFN-γ and TNF-α) showed global effects on induction of these chemokines. IL-1β increased MDC, MCP-1 and MIP-1α by 1·5-, 2·5- and 3·5-fold, respectively. Similarly, TNF-α increased MDC, MCP-1 and MIP-1α by three-, two- and fourfold, respectively. TNF-α also increased IP-10 by twofold. However, IFN-γ only increased MDC (4·5-fold) and IP-10 (fivefold), but not MCP-1 and MIP-1α in SV-HUC-1 cells. SV-HUC-1 cells were also found to produce spontaneously high levels of IL-6 and IL-8 in culture. IL-6 was further up-regulated by IL-1β and TNF-α by two- and 2·5-fold, respectively. Similarly, IL-8 was up-regulated by IL-1β and TNF-α by twofold for each.

Fig. 3.

Fig. 3

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Chemokine production by human urothelial cell lines in response to bacillus Calmette–Guérin (BCG) or BCG-derived cytokines. SV-HUC-1, RT4 and T24 cells were cultured in the presence of BCG (0·01 OD/ml), rIL-1β (1 ng/ml), rIFN-γ (10 ng/ml) or rTNF-α (10 ng/ml) for 3 days. Conditioned media were then collected for enzyme-linked immunosorbent assay (ELISA) analysis of macrophage-derived chemokine (MDC), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), interferon-inducible protein (IP) and eosinophil chemoattractant activity (eotaxin), interleukin (IL)-6 and IL-8. Eotaxin was undetectable. *Significantly increased compared with non-treated cell culture (spontaneous secretion).

In contrast to normal urothelial SV-HUC-1 line, both bladder TCC lines RT4 and T24 showed severe defects in chemokine production. RT4 cells were found to produce no MCP-1 and MIP-1α, whereas T24 cells were found to produce no MDC and MIP-1α (Fig. 3). However, similar to SV-HUC-1 cells, both RT4 and T24 cells were responsive to BCG-derived cytokines (IL-1β, IFN-γ and TNF-α) for production of the remaining chemokines tested. As observed, IL-1β increased MDC in RT4 cells (15-fold) and IP-10 in T24 cells (twofold). Similarly, IFN-γ increased MDC in RT4 cells (16-fold) and IP-10 in both RT4 and T24 cells (19- and 18-fold, respectively). TNF-α increased MDC in RT4 cells (28-fold), MCP-1 in T24 cells (2·5-fold) and IP-10 in both RT4 and T24 cells (five- and 2·5-fold, respectively). In addition to the defects in MCP-1 and MIP-1α production, RT4 cells also produced no IL-6 and a much lower level of IL-8 than those of SV-HUC-1 and T24 cells. However, production of IL-8 in RT4 cells could be up-regulated by IL-1β and TNF-α (three- and 14-fold, respectively). As reported previously [30–33], T24 cells produced spontaneously high levels of IL-6 and IL-8 in culture. Like SV-HUC-1 cells, T24 cells produced increased IL-6 in response to IL-1β and TNF-α (three- and 3·5-fold, respectively). However, their IL-8 production was almost saturated under the experimental setting. Nevertheless, collectively, these results suggest that urothelial cells, even in malignancy, are probably responsive to BCG and/or its derived cytokines for chemokine production in vivo and may participate actively in the process of anti-tumour immunity induced by intravesical BCG immunotherapy.

PBMC chemokine production in response to BCG stimulation

To assess chemokine production by immune cells in response to BCG, PBMCs were prepared from both BCG-naive and BCG-vaccinated healthy subjects and cultured in the presence of BCG for 1 h for up to 7 days. At various time-points, culture supernatants were collected for ELISA analysis of CC- and CXC-chemokines, including MDC, MCP-1, MIP-1α, IP-10 and eotaxin. For comparison, culture supernatants were also analysed for cytokines IL-6, IL-10, IFN-γ, TNF-α and GM-CSF. BCG efficiently induced MDC, MCP-1, MIP-1α and IP-10, but not eotaxin, from PBMCs of both BCG-naive (Fig. 4) and BCG-vaccinated (Fig. 5) subjects. Among these chemokines, MCP-1 and MIP-1α appeared to be early chemokines, which were detectable as early as 4–5 h post-BCG exposure and peaked at day 1 for MCP-1 and days 1–4 for MIP-1α. IP-10 appeared to be an intermediate chemokine, which elevated at day 1 and peaked at day 2. MDC appeared to be a late chemokine, which was also elevated at day 1 but peaked at day 7. This kinetic pattern was paralleled with that of BCG-induced cytokines (early cytokine: TNF-α appeared at 3 h and peaked at days 1–2; intermediate cytokines: IL-6 and IL-10 appeared at 5–6 h and peaked at day 7 for IL-6 and day 4 for IL-10; and late cytokines: IFN-γ and GM-CSF appeared at day 1 and peaked at days 3–7). Although BCG-vaccinated subjects showed higher levels of IP-10, IL-10, IFN-γ and TNF-α (three-, two-, 35- and threefold, respectively), both BCG-naive and BCG-vaccinated subjects showed similar kinetics for chemokine and cytokine production. Other chemokines (MDC and MIP-1α) and cytokines (IL-6 and GM-CSF) showed comparable levels between BCG-naive and BCG-vaccinated subjects. Interestingly, BCG-naive subjects showed a higher level of MCP-1 than BCG-vaccinated subjects. These results indicate that BCG is a potent inducer for CC- and CXC-chemokine production from human mononuclear cells.

Fig. 4.

Fig. 4

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Bacillus Calmette–Guérin (BCG)-induced chemokine/cytokine production from peripheral blood mononuclear cells (PBMCs) of a BCG-naive healthy person. PBMCs were prepared and cultured in the presence of BCG (0·01 OD/ml) for 1 h up to 7 days. Conditioned media were harvested at the indicated time-points and processed for enzyme-linked immunosorbent assay (ELISA) analysis of chemokines [macrophage-derived chemokine (MDC), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), interferon-inducible protein (IP) and eosinophil chemoattractant activity (eotaxin)] and cytokines [interleukin (IL)-6, IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and granulocyte–macrophage colony-stimulating factor (GM-CSF)]. Minimal chemokines and cytokines were detected in unstimulated cultures (data not shown). Eotaxin was undetectable.

Fig. 5.

Fig. 5

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Bacillus Calmette–Guérin (BCG)-induced chemokine/cytokine production from peripheral blood mononuclear cells (PBMCs) of a BCG-vaccinated healthy person. PBMCs were stimulated with BCG and analysed by enzyme-linked immunosorbent assay (ELISA) for chemokine/cytokine production as described in Fig. 4. Minimal chemokines and cytokines were detected in unstimulated cultures (data not shown). Eotaxin was undetectable.

Discussion

In this study, we investigated human chemokine production in response to BCG stimulation from three distinctive sources, i.e. the bladder wall, urothelial cell lines and PBMCs. Our results demonstrate that BCG is a potent inducer of human chemokines MDC, MCP-1, MIP-1α, IP-10 and eotaxin. Our results also suggest that the BCG's anti-bladder cancer activity may depend on production of the investigated chemokines.

Currently, only a few human chemokines have been demonstrated to be responsive to BCG stimulation in vivo. Reale et al. reported elevated serum MCP-1 and regulated upon activation normal T cell expressed and secreted (RANTES) in bladder cancer patients after intravesical BCG instillation [34]. We and others observed elevated urinary IL-8 and IP-10 in BCG-treated bladder cancer patients [20–24]. In this study, we extended these observations and found that intravesical BCG instillation induced urinary CC-chemokines MDC, MCP-1, MIP-1α and eotaxin, other than CXC-chemokines IL-8 and IP-10, in BCG-treated bladder cancer patients. Because eotaxin was detected in neither mononuclear cells (Figs 4 and 5) nor urothelial cell lines (Fig. 3), the source of urinary eotaxin remains for further investigation.

BCG-induced urinary chemokines appeared to have no correlation with a favourable BCG response, as patients who failed BCG therapy also produced urinary chemokines. There was also no clear time-dependent chemokine release relative to each other in the urine. This phenomenon was due probably to the fact that multiple cellular sources in the bladder could contribute to urinary chemokines including resident immune cells, infiltrating inflammatory leucocytes and normal and residual malignant epithelial cells. Recently, detrusor smooth muscle cells were also found to be capable of producing certain chemokines and cytokines in response to inflammatory mediators [35–37]. In contrast to urinary chemokines, urinary IFN-γ probably served as a useful surrogate marker for BCG responders, as reported previously [19,25,26].

Several studies demonstrated that human TCC cell lines in culture were capable of producing certain CXC-chemokines (IP-10 and IL-8) and cytokines (IL-1β, IL-6, TNF-α, GM-CSF and IFN-α) either spontaneously or after stimulation with BCG or certain proinflammatory cytokines [21,30–33,38,39]. These observations suggest that bladder epithelial cells could secrete chemokines and cytokines into the urine after intravesical BCG immunotherapy. To determine further whether human urothelial cells could also produce CC-chemokines MDC, MCP-1, MIP-1α and eotaxin, we made use of three urothelial cell lines reflecting both normal and malignant status, i.e. SV-HUC-1 (a normal urothelial line), RT4 (a well-differentiated TCC line) and T24 (a poorly differentiated TCC line). Among all chemokines tested, including CXC-chemokines IP-10 and IL-8, eotaxin was the only chemokine undetectable in all three cell lines. Chemokine production appeared to be intact in the SV-HUC-1 line but defective in both the RT4 and T24 lines, as no MCP-1 and MIP-1α were found in the RT4 line and no MDC and MIP-1α were found in the T24 line, respectively (Fig. 3). In addition, the RT4 line also produced no IL-6 and a low level of IL-8. This observation indicates that the chemokine/cytokine machinery is altered in malignant urothelial cells and that this alteration is heterogeneous. Although BCG appeared to have no or marginal effects on induction of chemokines tested under experimental settings, BCG-derived cytokines (IL-1β, IFN-γ and TNF-α) found in the urine after intravesical BCG immunotherapy showed global effects on promoting chemokine production. IL-1β increased MDC, MCP-1, MIP-1α and IL-8, whereas TNF-α increased these chemokines plus IP-10 in SV-HUC-1 cells. IFN-γ also increased MDC and IP-10 in SV-HUC-1 cells. Although both RT4 and T24 cells lacked an ability to produce certain chemokines, they were responsive to IL-1β, IFN-γ and TNF-α and produced MDC, IP-10 and IL-8 in RT4 cells and MCP-1, IP-10 and IL-8 in T24 cells, respectively. Thus, it is likely that through the induction of proinflammatory cytokines BCG affects both normal and malignant urothelial cells to produce chemokines during intravesical BCG immunotherapy.

We have observed a clear kinetic pattern for BCG-induced chemokines from human PBMCs (early: MCP-1 and MIP-1α; intermediate: IP-10; and late: MDC). This kinetic pattern was paralleled with that of BCG-induced cytokines (early: TNF-α; intermediate: IL-6 and IL-10; and late: IFN-γ and GM-CSF). PBMCs of both BCG-naive and BCG-vaccinated subjects showed similar kinetics, although the latter PBMCs expressed higher levels of certain chemokines and cytokines. This phenomenon suggests that chemokine/cytokine production in BCG-stimulated mononuclear cells is temporally regulated, a process reflecting the complex interactions between these two types of proinflammatory mediators [3,40]. The PBMC chemokine production observed may be a secondary event to cytokine production by these cells in response to BCG, as exogenous IFN-γ and TNF-α were found to be capable of inducing chemokines from PBMCs including MDC, MCP-1 and IP-10 (data not shown).

Among the chemokines detected in PBMC cultures, MDC showed a remarkably high level compared with MCP-1, MIP-1α and IP-10. Because MDC appeared to be a late chemokine, its high expression was probably attributed to the collective actions of early chemokines and cytokines in the same cultures. Indeed, MCP-1 and MIP-1α are known to be potent activators of monocytes/macrophages [41,42], a major cellular source of MDC [43]. Cytokines GM-CSF, IFN-γ and TNF-α are also capable of stimulating monocytes/macrophages [40]. In contrast to MDC, eotaxin was undetectable in BCG-stimulated PBMC cultures. This observation was consistent with the known cellular sources for eotaxin. It has been reported that eotaxin is produced mainly by epithelial cells, endothelial cells and fibroblasts but not by leucocytes [44,45].

It is generally accepted that the BCG-induced anti-tumour immunity is dominated by a localized non-specific immune response involving development of various proinflammatory chemokines and cytokines. The results of the present study demonstrate that BCG is a potent inducer of human CC- and CXC-chemokines. Based on the current understanding of BCG action [7–9,46], bladder epithelial cells, including residual tumour cells, act as the first-line cells to produce chemokines and cytokines when encountering intravesically instilled BCG. The locally residing dendritic cells and macrophages may also produce chemokines and cytokines upon activation after phagocytosis of BCG at early stage. These early produced chemokines and cytokines can cause infiltration and activation of inflammatory leucocytes as well as activation of resident immune cells and detrusor smooth muscle cells to produce more chemokines and cytokines, leading subsequently to a profound cellular response inside the bladder. Thus, BCG-induced chemokine/cytokine responses may represent one of the mechanisms by which BCG exerts its anti-tumour activity. Further studies will be necessary to characterize the complex interaction between these chemokines and cytokines and the key mediator(s) responsible for developing BCG-induced anti-tumour immunity in intravesical BCG immunotherapy.

Acknowledgments

This work was supported in part by grants from the National Institutes of Health (RO1DK66079) and Department of Defense (W81XWH-04-1-0070). We would like to thank Mitchell L. Rotman for helping to edit the manuscript.

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