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Impaired monocyte function in cancer patients: restoration with a cyclooxygenase-2 inhibitor¹

Department of Otorhinolaryngology, Head and Neck Surgery and
^* Institute for Medical Informatics, Biometry, and Epidemiology at the Ludwig-Maximilians-University of Munich, Munich D-81377, Germany; and
Vaecgene Biotech, Munich D-81377, Germany

²Correspondence: Department of Otorhinolaryngology, Head and Neck Surgery, Grosshadern Medical Center; Ludwig-Maximilians-University of Munich, Marchioninistr. 15; 81377 Munich, Germany. E-mail: stlang{at}hno.med.uni-muenchen.de

SPECIFIC AIMS

Carcinoma patients suffer from a tumor-suppressed immune system. This phenomenon is attributable to the tumor itself, which produces and secretes immunosuppressive factors such as prostaglandin E2 (PG), known to be elevated in cancer patients. Cyclooxygenase (COX) inhibitors that block PG synthesis demonstrated remarkable anti-cancer effects. Evidence has accumulated showing that inhibition of COX-2 plays a key role in suppressing multistep carcinogenesis. However, the exact mechanism as to how NSAIDs prevent cancer is still unclear.

PRINCIPAL FINDINGS

1. CCR5 expression is reduced on monocytes isolated from tumor patients
Proper expression of chemokine receptors is a pivotal prerequisite for the extravasation of monocytes, and thus for physiological function. We recently demonstrated that the chemokine receptor CCR5 is down-regulated on monocytes after incubation of these cells in PG-containing supernatants from carcinoma cell lines. We have also shown that this receptor was not down-regulated when these supernatants were generated in the presence of NSAIDs, namely, aspirin or indomethacin. Since squamous cell carcinomas of the head and neck (SCCHN) overexpress COX-2 and consequently produce high amounts of PG, we investigated CCR5 expression levels and the migration capacity of monocytes from tumor patients vs. healthy donors in vivo.

PBMCs were isolated from SCCHN patients and healthy volunteers and were analyzed by flow cytometry. This first series of investigations revealed that monocytes from SCCHN patients (n=31) displayed significantly lower levels of surface CCR5 than those seen in healthy donors (n=40, P=0.02).

We asked whether soluble factors in the patients’ sera accounted for this suppressive effect. We incubated monocytes from healthy donors for 1 day in allogeneic sera from either SCCHN patients or healthy volunteers. These monocytes expressed significantly less CCR5 (P=0.0266) after incubation in allogeneic SCCHN sera (2.88±0.75; n=8) compared with their incubation in healthy sera (3.90±0.98; n=10). In conclusion, sera from tumor patients contain immunosuppressive factors that down-regulate CCR5 on monocytes.

2. Cyclooxygenase inhibition restores monocyte function
Considerable evidence supports the antineoplastic effect of NSAIDs. However, this effect is not well understood on the molecular level, and COX-dependent as well as COX-independent mechanisms have been described. Hence, cells expressing cyclooxygenase (especially COX-2), including tumor cells, are potential targets for NSAIDs.

To test this hypothesis, we initiated a clinical study with tumor patients suffering from SCCHN. Patients enrolled in this study received the COX-2-inhibiting drug Rofecoxib (n=15) for 3 wk before surgery and showed no side effects due to the drug. Patients of the control group (n=13) were left untreated. Monocytes from each patient were analyzed for CCR5 expression before (=day 0) and after this period (=day 21). Much to our surprise, within these 3 wk we observed a significant down-regulation of the CCR5 receptor on monocytes in the untreated control group (P=0.0273; Fig. 1 ), whereas CCR5 expression increased (P=0.0027) in the Rofecoxib-treated cancer patients to levels significantly higher at the end of the treatment period (day=21) than in the untreated control group (P=0.0011).

View larger version (20K): [in this window] [in a new window]   Figure 1. Rofecoxib improves CCR5 expression on monocytes. Percent change from base line of CCR5 expression on monocytes from SCCHN patients who were treated with Rofecoxib (25 mg/day) or left untreated (control). Base lines at day = 0 were set at 100% for each patient enrolled in this study. Horizontal lines show the mean changes. P values for comparison within a group are based on the Wilcoxon test; the nonparametric U test was used for intergroup comparison.

To test the immunosuppressive effects of tumor patients’ sera, we incubated monocytes from healthy donors for 1 day with sera derived from cancer patients before (day=0) and after (day=21) the oral intake of Rofecoxib for 3 wk preoperatively. Control patients were left untreated. Mean CCR5 values at day = 0 were set as the base line. At day 21, sera from control patients (n=8) more strongly suppressed CCR5 expression on healthy monocytes than did sera from patients of the Rofecoxib group (n=10; P=0.0014). Moreover, sera from patients of the control group were more suppressive at day = 21 than at day = 0 (P=0.0059). In contrast, the suppressive effect was significantly reversed in the Rofecoxib group (P=0.0142) within the same period (data not shown).

3. Rofecoxib improves the migration of monocytes from tumor patients
A prerequisite for proper immune function is the high degree of cell motility during immune responses. Cell migration is a complex process that includes chemoattraction and extravasation. Since we observed reduced expression of CCR5 on monocytes from tumor patients, we investigated the potential of these cells to migrate in the direction of a chemotactic stimulus, namely, MIP-1ß. To do this, we conducted chemotaxis assays with primary monocytes isolated from patients with SCCHN or from healthy donors. Migration was performed toward MIP-1ß (20 ng/mL) for 45 min through 8 µm pore filters. As pointed out in Fig. 2 , a significantly (P=0.0007) lower number (2.36%±3.40; n=16) of monocytes from SCCHN patients migrated compared with those from healthy donors (16.9%±14.1; n=6). The intake of Rofecoxib for 21 days improved migration (13.4%±12.3; n=10) almost to the levels of healthy persons. During the same period, the number of migrated monocytes from untreated tumor patients further declined to nearly undetectable levels (0.23%±0.41; n=6; data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 2. The migration capacity of monocytes, impaired in SCCHN patients (=day 0), can be significantly restored to almost normal levels (=healthy donors) by administration of Rofecoxib for 21 days.

CONCLUSIONS AND SIGNIFICANCE

The COX pathway recently raised the interest of oncologists when COX-2 was found to be overexpressed in various tumors (7, 16). Since then, multiple experimental systems and epidemiological studies have demonstrated that long-term use of NSAIDs reduces the risk of cancer remarkably (7, 9). Most important, specific COX-2 inhibitors such as Rofecoxib show promise as chemopreventive agents. The molecular mechanism underlying the antineoplastic effect of NSAIDs is not yet well understood, but may include their ability to inhibit prostaglandin synthesis.

In a clinical study with patients suffering from head and neck cancer, we observed significant suppression of chemokine receptor CCR5 expression and an impairment in monocyte migration capacity of monocytes compared with cells from healthy volunteers. Administration of Rofecoxib as a specific COX-2 inhibitor for 3 wk completely restored CCR5 expression and monocyte migration. Such a restoration in immunity is usually observed only after surgical removal of the tumor. Since COX-2 overexpression along with prostaglandin production can be found in various types of cancer, inhibition of this enzyme may contribute to the antineoplastic effects of NSAIDs.

Impairment of the immune system is a common phenomenon in tumor patients. Our data provide evidence that this immunosuppression is due at least in part to soluble factors produced by the tumor itself, since sera from tumor patients displayed a suppressive effect on monocytes from healthy donors. We emphasize that this down-regulation is a consequence of COX-overexpression, probably within the tumor, since it was reversible by a COX-2 inhibitor.

The data presented here are to the best of our knowledge the first to demonstrate a selective COX-2 inhibitor’s immunorestorative effect in vivo and provide the rationale for the use of NSAIDs for chemoprevention or immunoadjuvant cancer therapies. We demonstrate that elevated COX-2 expression in cancer patients can no longer be considered a simple epiphenomenon of carcinogenesis, but rather as an active contributor to host immunosuppression. Taken together, we describe systemic effects of a selective COX-2 inhibitor on a whole class of immune effector cell in tumor patients. These data contribute to our understanding of the anti-tumor effect of these drugs at the molecular and functional level, and may help to improve the clinical prognosis of cancer patients.

View larger version (18K): [in this window] [in a new window]   Figure 3. Tumor cells overexpress COX-2 and produce high amounts of PG, resulting in down-regulation of CCR5 and the impaired migration of monocytes. Inhibition of PG synthesis by NSAIDs such as aspirin, indomethacin, and Rofecoxib restores these parameters in tumor patients to practically normal levels.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0417fje; to cite this article, use FASEB J. (December 18, 2002) 10.1096/fj.02-0417fje

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