Published online before print August 21, 2003
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enna Mytar
oszyn Szatanek
ckiewiczDepartment of Clinical Immunology, Polish-American Institute of Pediatrics, Jagiellonian University Medical College, Cracow, Poland
1 Correspondence: Department of Clinical Immunology, Polish-American Institute of Pediatrics, Jagiellonian University Medical College, Wielicka 265, 30-663 Cracow, Poland. E-mail: mizembal{at}cyf-kr.edu.pl
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, TNF; IL-10; IL-12p40) and cytotoxicity, is altered by exposure to cancer cells. Tumor cell-pre-exposed monocytes restimulated with tumor cells showed significantly decreased production of TNF, IL-12, increased IL-10 (mRNA and release) and inhibition of IL-1 receptor-associated kinase-1 (IRAK-1) expression. This down-regulation of cytokine production was selective, as the response of pre-exposed monocytes to lipopolysaccharide (LPS) was unaffected. Treatment of tumor cell-pre-exposed monocytes with hyaluronidase (HAase) improved their depressed production of TNF, while HAase-treated cancer cells did not cause monocyte dysfunction. The response of hyaluronan (HA)-pre-exposed monocytes to stimulation with tumor cells was also inhibited. Cytotoxic activity of monocytes pretreated with cancer cells was also decreased. This study shows that tumor cells selectively deactivate monocytes and suggests that tumor cell-derived HA by blocking CD44 on monocytes inhibits their antitumor response. These observations may provide some explanation for the depressed function of TIM in human malignancy.
Key Words: cytokines • hyaluronan • IRAK-1
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Monocytes/macrophages are involved in antitumor response of the host and act both as cells presenting tumor-associated antigens to tumor-infiltrating lymphocytes and as cytotoxic effector cells. In comparison to peripheral blood monocytes that exhibit a significant spontaneous cytotoxicity against malignant cells [1
, 2
], antitumor activity of tumor infiltrating macrophages (TIM), including interleukin 12 (IL-12) production, is depressed in cancer patients [3
, 4
]. This may suggest that the local tumor microenvironment adversely modifies the functional activity of TIM in situ. There is also evidence that during the growth of experimental tumors suppression of the function of local, but not distant, macrophages occurs [5
]. Multiple mechanisms may be involved, for example, production of various mediators by tumor cells that down-regulate cytotoxic potential of TIM. Cytokines (IL-4, IL-10, transforming growth factor ß - TGFß) or prostaglandins (PGE2) produced by tumor cells inhibit production by mononuclear cells of interferon gamma, tumor necrosis factor (TNF), IL-12, reactive oxygen intermediates (ROI), and reactive nitrogen intermediates (RNI) that possess proinfammatory and antitumor properties [5
6
7
]. This unresponsiveness (or deactivation) is unrestricted as monocytes not only do not respond to tumor cells but also other activators, like the Staphylococcus aureus Cowan strain 1 [7
].
We have previously shown that cancer cells induce production of cytotoxic molecules (TNF, ROI, RNI) by monocytes [2 , 8 , 9 ] and provided evidence for the involvement of CD44 molecule, the major receptor for hyaluronan (HA), in signaling for their generation [10 ]. HA, a high-molecular-weight glycosaminoglycan, is involved in the maintenance and hydration of matrix structure, cell proliferation, differentiation, and locomotion [11 ], inflammation and wound healing [12 ]. HA is produced and shed in abundant quantities by many tumor cells [13 ], and its enhanced expression is associated with their metastatic potential [14 ]. Recently, HA has received considerable attention because of its growth-promoting effect in human malignancy and the association of strong HA expression on cancer cells with the unfavorable prognosis [15 ]. We have recently suggested that HA on cancer cells is an important molecule in the induction of ROI generation by monocytes, but free HA may inhibit monocyte interactions with cancer cells [10 ]. Siegert et al. [16 ] showed suppression of ROI production by human macrophages cocultured with colon adenocarcinoma cell lines that was not mediated by IL-4, IL-10, and TGFß1.
It has been recently hypothesized that cancer progression is an inherently proinflammatory process that activates innate and adaptive antitumor immunity. To compromise immune surveillance, cancer cells must develop mechanisms that inhibit proinflammatory signals, thereby shifting balance from activation to unresponsiveness [17
, 18
]. Inflammation may be triggered when specific receptors, known as pattern receptors (for example, Toll-like receptors—TLR2, TLR-4, etc.) recognize non-self patterns (carbohydrate structures, phosphatidylserine) present on tumor cells. Following recognition, the receptors bind to an intracellular adaptor proteins, like myeloid differentiation factor 88 (MyD88), which recruits IL-1R-associated kinase-1 (IRAK-1) to this receptor-signaling complex. IRAK-1 is then phosphorylated and activated and leaves the membrane-bound receptor complex, associates with TNF receptor-associated factor 6 (TRAF6) and activates downstream-signaling pathways such as mitogen-activated protein (MAP) kinases and the transcription factor NF-
B. NF-B is then translocated to the nucleus and activates transcription of the major proinflammatory cytokines: TNF, IL-12p40, IL-18 [19
20
21
]. In such NF-B-activating signal transduction cascade, IRAK-1 seems to play a key role, as it is down-regulated after triggering with proinflammatory stimuli known to induce immunological tolerance [22
, 23
].
The present studies were designed to investigate whether and how antitumor activity of monocytes may be altered by contact with tumor cells. The in vitro model of in situ TIM-tumor interactions was used in which human blood monocytes are cocultured with tumor cells. The data show that contact with tumor cells leads to a selective deactivation of monocyte function (supressed production of TNF and IL-12 in response to restimulation with tumor cells, but not LPS, and depressed cytotoxic activity), which is associated with down-regulation of IRAK-1 expression. Tumor cell-derived HA may be an important molecule in inhibition of proinflammatory cytokines production by monocytes.
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Cell lines
The following human cell lines were used: HPC-4 (pancreatic adenocarcinoma), DeTa (colorectal adenocarcinoma) and HCV29 (normal uroepithelium) described previously [10
]. A549 (lung carcinoma), Jurkat (T cell leukemia) and Raji (Burkitt lymphoma) were provided by Dr. D. Du
(Institute of Immunology, Wrocaw, Poland). CHP100 (neuroblastoma), RD3 (rhabdomyosarcoma), and HTB26 (breast cancer) were obtained from Professor M. Z. Ratajczak (Cancer Center, Louisville, KY). HAE (normal bronchial epithelium) was a kind gift from Dr M. Frankenberger (Gauting, Munich, Germany). Freshly isolated breast carcinoma cells were provided by Dr. J. Trojan (1st Department of Surgery, Jagiellonian University Medical College, Cracow, Poland). Cells were cultured by biweekly passages in RPMI 1640 with 5% FCS. Cell lines were regularly tested for Mycoplasmasp. contamination by polymerase chain reaction (PCR) ELISA test (Roche Diagnostics GmbH, Mannheim, Germany) according to manufacturer’s protocol.
Cell cultures
Isolated monocytes were cultured either in the medium (control) or with tumor cells at the ratio 1:0.3 for 3 h (pre-exposure). After staining with phycoerythrin-labeled anti-CD14 (Becton-Dickinson, San Jose, CA) monoclonal antibody (mAb), CD14+cells were sorted out with FACS Vantage Cytometer (Becton-Dickinson) equipped with a Power Macintosh 7600/120 computer using a Cell Quest v. 3.0 software (for details, see [2
]). The ion laser Innova Enterprice II (Coherent, Santa Clara, CA) operating at 488 nm was used as a light source. After setting CD14+ (monocytes) and CD14- (tumor cells) gating, sorting was performed with the use of a 70 µm nozzle tip and a drop drive frequency of 25 kHz, three-drop envelopes and "normal-R" sort mode. Sorted cells were collected into a water-cooled (constant temperature circulator, Neslab Instruments, Inc., Portsmouth, NH) polystyrene Falcon 2057 tubes (Becton-Dickinson) precoated with FCS to avoid cell attachment. The purity of sorted cells was checked by flow cytometry, and it exceeded 98%. Isolated CD14+ monocytes were cultured alone, with tumor cells (HPC-4 or DeTa) or lipopolysaccharide (LPS, Sigma, St. Louis, MO; 100 ng/ml) in flat-bottom 96-well microtiter plates (Nunc, Roskilde, Denmark) for 18 h in a humidified 5% CO2 atmosphere. As a control, CD14+ cells were sorted out from monocytes cultured in the medium alone ("dummy sorting") or with normal epithelial HCV29 cells. In some experiments, CD14+ monocytes were pre-exposed to LPS (20 µg/ml or 100 ng/ml), washed three times, and then stimulated either with tumor cells or LPS (100 ng/ml). Then supernatants were collected and tested for cytokine content. In some experiments, monocytes were preincubated with HA (Sigma, 100 µg/ml, endotoxin level <30 pg/mg).
Determination of cytokines
Concentrations of TNF, IL-10, and IL-12p40 in the culture supernatants were measured by appropriate ELISA kits (PharMingen, San Diego, CA), according to the manufacturer’s instruction. Detection level for TNF was 20 pg/ml, for IL-10 and IL-12 10 pg/ml.
Determination of cytokine mRNA expression by LightCycler RT-PCR
The total RNA was extracted from monocytes by the single-step isolation method using TRI-ZOL reagent (Gibco-BRL, Grand Island, NY) according to the manufacturer’s protocol. The first-strand cDNA was obtained from the total RNA samples (2 µg) with M-MLV reverse transcriptase (Sigma) and oligo-dT (Gibco) primer as specified by the manufacturer’s protocol. The quantitative PCRs for TNF-, IL-10, and IL-12p40 were performed using the LightCycler system (Roche Diagnostics, Mannheim, Germany). The following primer pairs were used for TNF: sense, 5'-CAG-TCA-GAT-CAT-CTT-CTC-GA, and antisense, 5'-TCA-CAG-GGC-AAT-GAT-CCC-AAA; for IL-10: sense, 5'-GGA-CTT-TAA-GGG-TTA-CCT-GG, and antisense, 5'-GAA-CTC-CTG-ACC-TCA-AGT-GA; and for IL-12p40: sense, 5'-AAC-TGG-ACC-TTG-CAC-CAG-AG, and antisense, 5'-AGA-CTC-TCC-TCA-GCA-GCT-GG. In brief, 3 µl of the cDNA were used for each quantitative LightCycler PCR run, using the Light-Cycler-DNA Master SYBR Green I kit from Roche (Mannheim, Germany). Amplifications were carried out in the total volume of 20 µl with the final MgCl2 concentration of 3 mM, and 0.5 µM of each primer. Each LightCycler PCR run consisted of 45 cycles with initial denaturation time of 5 min. at 95°C. For TNF-, the cycling profile was set at 95°C for 0 s., 62°C for 10 s., 72°C for 19 s.; for IL-10, 95°C for 0 s., 62°C for 10 s., 72°C for 40 s.; and for IL-12p40, 95°C for 0 s., 60°C for 25 s., and 72°C for 40 s. The fluorescent signals generated during the informative log-linear phase were used to calculate the relative amount of mRNA. The melting curve analysis was performed to verify the specificity of the amplified products. The mRNA expression is indicated as a fold difference in comparison to unstimulated control monocytes.
Enzyme treatment
Monocytes or tumor cells at 1 x 106/ml were treated with hyaluronidase (HAase, from bovine testes, Sigma, 100 U/ml) or sialidase (neuraminidase from Vibrio cholerae, Sigma, 0,04 U/ml) for 2 h at 37°C with gentle shaking [25
]. The reaction was terminated by washing. The treatment did not affect cell viability.
Western blotting
Cytoplasmatic protein extracts separated as described previously [26
] were loaded with 10 µg/lane, on 4-12% Tris-glycine gels (EC60385, Invitrogen, Karlsruhe, Germany) followed by electroblotting using Xcell SureLock Mini-Cell and Xcell II Blot Module (E10002; Invitrogen). Blots were incubated with anti-IRAK-1 mAb (sc-5287) (Santa Cruz Biotechnology, Santa Cruz, CA) followed by goat anti-mouse peroxidase-conjugated Ab (Sigma), developed with enhanced chemiluminescence reagent (RPN2106; Amersham, Braunschweig, Germany), and exposure to Hyperfilm (RPN3103; Amersham). The specificity of staining was confirmed using as a positive control appropriate cytoplasmic protein extracts provided by the manufacturer. Blots were scanned and then analyzed using the analySIS program (Soft Imaging System, Münster, Germany).
Cytotoxicity assay
Monocyte cytotoxicity was tested according to the method described previously [2
]. Briefly, control monocytes (5 x 104/well) or monocytes pre-exposed to HPC-4 or DeTa cancer cells were cultured either with the same cancer cells (2 x 104/well) or in a criss-cross manner for 18 h. Then, the culture medium was removed and 100 µl of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 2 mg/ml, Sigma) dye solution was added. Cell proliferation was assessed by reduction of MTT. Data were expressed as the percentage of cytotoxicity [2
].
Statistical analysis
Statistical analysis was performed by ANOVA. Differences were considered significant at p values < 0,05.
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Figure 1. Cytokine secretion by tumor cell-pre-exposed and control monocytes in response to stimulation with HPC-4 cancer cells or LPS. Monocytes exposed to HPC-4 cancer cells for 3 h (CD14+/tumor) were sorted out and cultured with HPC-4 cells or LPS (100 ng/ml). As control monocytes kept in the medium or cultured with normal epithelial cells were also sorted (CD14+/medium and CD14+/normal, respectively) and then stimulated with either cancer cells or LPS. The level of cytokines released by unstimulated control monocytes was <20 pg/ml and by pre-exposed monocytes in the range 0-420 pg/ml for TNF, 0-54 pg/ml for IL-12 and 0-155 pg/ml for IL-10. Data based on 8-12 different experiments. Mean ± SE is shown. **P < 0.005, *P < 0.05.
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Figure 2. Cytokine secretion by LPS-pre-exposed and control monocytes in response to stimulation with HPC-4 cancer cells or LPS. CD14+ monocytes were preatreated for 3 h with 20 µg/ml or 100 ng/ml of LPS (CD14+/LPS20µg or CD14+/LPS100ng, respectively) or kept in the medium (CD14+/medium). After washing, monocytes were stimulated with either tumor cells or LPS (100 ng/ml) and cultured for 18 h. Data based on 3 independent experiments. Mean ± SE is shown. *P < 0.05 when compared with control monocytes.
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Table 1. Cytokine Secretion by Monocytes Stimulated with Different Cell Lines or Pre-exposed to Different Tumor Cell Lines and Stimulated with HPC-4 Cells
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-enolase gene were comparable (data not shown).
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Figure 3. mRNA expression of TNF, IL-12p40, and IL-10 in control (A) and cancer (HPC-4) cell-pre-exposed (B) monocytes stimulated with the same cancer cells. Monocytes were cultured in the medium (control) or with HPC-4 cells for 2 h, then CD14+ cells were sorted out and stimulated with HPC-4 cells for 1 h (TNF), 3 h (IL-10) and 4 h (IL-12). To control monocytes HPC-4 cells were added at the end of culture. Following isolation of RNA, RT and real-time monitoring of cytokine-mRNA using the LightCycler technology was performed. Average cytokine-mRNA is expressed in relative units (fold difference in comparison to unstimulated control monocytes). Mean ± SD of 3 independent experiments is shown.
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70% in cancer cell-pre-exposed monocytes, whereas in normal epithelial cell-pre-exposed monocytes, it remained unchanged (Fig. 4
).
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Figure 4. Expression of IRAK-1 in tumor cell-pre-exposed and control monocytes. Elutriated monocytes from healthy donors were co-cultured with tumor (CD14+/tumor) or normal cells (CD14+/normal) for 3 h, the cells were then stained with anti-CD14-PE mAbs, and CD14+ monocytes were sorted out (CD14+/tumor and CD14+/normal, respectively). Control monocytes cultured with medium alone were also stained and dummy-sorted (CD14+/medium). Cytoplasmic extracts were prepared from monocytes, blotted and stained with anti-IRAK-1 mAbs (see upper panels; one representative experiment for three performed). Two separate experiments with tumor and normal cells were run because of the limited number of sorted CD14+ cells. Blots were analyzed by densitometry, and the average results are given in the lower panels (n = 3).
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Figure 5. TNF secretion by (A) control or (B) tumor cell-pre-exposed monocytes pretreated with HAase or Nase or both enzymes, stimulated with cancer cells. CD14+ cells cultured in the medium (control) or cocultured with HPC-4 cells for 3 h were isolated by FACS sorting and then left untreated (medium) or treated for 2 h with HAase (100 U/ml), Nase (0,04 U/ml) or both enzymes. After washing, CD14+ monocytes were admixed to HPC-4 cells and cultured for 18 h. The results of 4 different representative experiments are shown. Mean ± SE is shown. Significantly enhanced in comparison to untreated control (medium) *P < 0.05.
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Figure 6. TNF secretion by monocytes pre-exposed to HAase- and Nase-pretreated cancer cells. HPC-4 cells were pretreated for 2 h with HAase (100 U/ml), sialidase (0,04 U/ml) or both enzymes, washed, and cocultured with monocytes for 3 h. Then, CD14+ monocytes from the control cultures and cocultures were isolated by FACS sorting and stimulated for 18 h with HPC-4 cells. Data are based on 5 different experiments. Mean ± SE is shown. Significant inhibition in comparison to control monocytes *P < 0.05.
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Figure 7. The effect of HA on TNF secretion by monocytes stimulated with tumor cells. (A) Fresh monocytes were either preincubated with HA (100 µg/ml) for 2 h and washed or (B) HA was added to monocytes that were then stimulated with HPC-4 cancer cells or with LPS. Data based on 4 different experiments are shown (mean ± SE). *P < 0.05
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Figure 8. Cytotoxic activity against cancer cells of control monocytes (CD14+/medium) or monocytes pre-exposed to HPC-4 (CD14+/HPC-4) or DeTa tumor cells (CD14+/DeTa). Sorted out CD14+ monocytes were then cultured with either HPC-4 or DeTa cells for 18 h, and cancer cell proliferation was assessed by the MTT test. The results of 7 experiments (mean±SE) are shown. *P < 0.05.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This in vitro model of monocyte-tumor cell interactions may be biologically relevant as mononuclear cells from patients with colorectal and breast cancer have defective IL-12 production, while generating an increased amount of IL-10 [30 , 31 ]. Also TIM from human ovarian cancer show enhanced IL-10 and defective IL-12 production. IL-10 acting in autocrine fashion is responsible for decreased IL-12 secretion [4 ], and it is likely that a similar mechanism may be involved in the present system. Also IL-10 and other immunosuppresive tumor-derived molecules (TGFß, IL-4, PGE2) may be involved in deactivation of monocytes [6 , 28 , 32 ], but it is not selective [32 ].
Tumor cell-pre-exposed monocytes showed the significant inhibition of IRAK-1 expression. It may be suggested that restimulation with tumor cells engages almost exclusively IRAK-1/NFB-dependent intracellular signaling pathway (for review, see [33
]), that is insufficient after previous contact with tumor cells. On the other hand, signals induced by LPS restimulation may bypass blocked IRAK-1 "checkpoint" by activating other receptors and signaling cascades rather than classical TLR4/IRAK-1/NFB signal transduction pathway leading to TNF and IL-12p40 gene expression (for review, see [34
35
36
]). Our other observations indicate that IRAK-1 degradation is not a major mechanism for induction of LPS tolerance (unpublished results). The mechanisms triggering IRAK-1 down-regulation in monocytes after contact with tumor cells are now investigated. The enhanced production of IL-10 by tumor-pre-exposed monocytes following restimulation with tumor cells may be explained by the fact that IL-10 gene expression is regulated mostly by Stat3 and is NFB-independent [37
]. It is possible that this enhanced synthesis of IL-10 is an additional factor in down-regulation of TNF and IL-12, as shown for IL-12 inhibition in TIM isolated from ovarian cancer that occurs via suppression of NFB-activation [4
]. The transcription factors regulating IL-10 synthesis after contact with tumor cells are also under investigation.
Our previous observations showed that CD44 molecules of monocytes are important for their interactions with cancer cells that result in TNF, RNI, and ROI production [2 , 24 , 38 ]. As CD44 is the major receptor for HA, we asked whether tumor cell-derived HA can block cellular interactions by binding to CD44. This was approached by studying the effect of sialidase and HAase. Sialidase, known to cause deglycosylation of CD44 by removal of sialic acid residues, increases binding of HA to CD44 [27 , 39 ], while HAase removes cell-associated HA [40 ]. Significantly compromised response of tumor cell-pre-exposed monocytes to restimulation, as measured by TNF release, was substantially increased following treatment with both HAase and sialidase. In contrast, response of control monocytes was unaffected by HAase and increased by sialidase. Therefore, effect of sialidase on control monocytes and monocytes from the coculture may be explained by their increased CD44-mediated ability to react with tumor cell-associated HA and further supports our previous observations on the role of CD44 in monocyte-tumor cell interactions [10 ]. HAase by removing cell-bound HA restores ability of deactivated monocytes to react with cancer cells. It is also in keeping with the effect of the enzymes on cancer cells used for the cocultures, as HAase-treated cancer cells did not cause monocyte deactivation, while sialidase had no effect. Also malignant T and B lymphoid cells, that do not synthesize HA [41 ], did not change monocyte function. Furthermore, when fresh monocytes were preincubated with HA and then stimulated with cancer cells, the release of TNF was significantly decreased. In contrast, the response to stimulation with LPS was unaffected. Altogether, these observations imply the role of HA in selective deactivation of monocytes.
It is known that HA is overexpressed on many tumor cells—in particular metastatic cells—and is shed from them sometimes in abundant quantity [14 , 42 ]. In fact, tumor stroma is enriched in HA [15 ]. Furthermore, we have observed that CD14+ monocytes isolated from the cocultures with tumor cells had an increased expression of CD44 variant isoforms v6 and v7/8 [43 ], which are known to be up-regulated by HA and to bind HA more avidly [44 , 45 ]. These data suggest that tumor-derived HA may block CD44 on monocytes, so they are unable to react efficiently with cancer cells. The role of other tumor cell-associated ligands for monocyte receptors and identification of intracellular signaling pathways involved in selective monocyte deactivation is under study.
This may have important biological implications for the activity of TIM in the tumor bed. Blood monocytes migrating from the circulation that possess substantial cytotoxic potential are deactivated in situ. By blocking CD44, HA interrupts interactions of monocytes with cancer cell-associated HA, mostly expressed in the synthase-bound form [46 ] and may be one of the tumor-derived signals responsible for the occurrence of IL-12- IL-10+ phenotype of TIM [4 ]. Clinicopathological observations indicate that strongly HA-positive tumors have a worse prognosis. Furthermore, the inverse correlation between the number of tumor-infiltrating lymphocytes and HA intensity on cancer cells suggests that HA may mask immunological recognition and inhibit lymphocyte recruitment [15 ]. In keeping, the present data implicate that tumor-derived HA dampens TIM’s ability to distinguish and react with tumor cells. Our unpublished observations indicate that the secretion of TNF and IL-12 by PBMC of patients with stage IV gastric cancer in response to stimulation with cancer cells is depressed. This may indicate that systemic deactivation of monocytes may also occur in patients with advanced cancer. Although it is not clear whether HA may also be involved, its serum level is often increased in patients with disseminated neoplasm [47 ].
In conclusion, the present data indicate that contact with tumor cells may cause monocyte dysfunction, which is associated with IRAK-1 down-regulation and induces TNF- IL-12- IL-10+ (M2) phenotype, characteristic for TIM [29 ].
Received April 7, 2003; revised July 4, 2003; accepted July 24, 2003.
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