CD169 (sialoadhesin) is a sialic acid receptor that is expressed on specific macrophages such as lymph node sinus macrophages. Animal studies have suggested that CD169+ macrophages have a pro-inflammatory property, however, the role of these cells in human diseases has not been clarified. In our in vitro experiments with human macrophages, pro-inflammatory cytokines, such as type 1 interferon, induced strong expression of CD169, suggesting that CD169 might be a specific marker of inflammatory macrophages. To examine the role of CD169 in antitumor immunity, we examined the expression of CD169 in regional lymph nodes (RLNs) and its association with overall survival in colorectal carcinoma (CRC). In a clinicopathological analysis on 83 CRC patients, paraffin-embedded specimens were evaluated for CD169 expression of RLN macrophages by immunohistochemistry. We found, for the first time, a high density of CD169+ macrophages was significantly associated with longer overall survival; multivariate analysis showed that the ratio of CD169+ cells to CD68+ cells was an independent prognostic factor. The majority of CD169+ macrophages were in direct contact with CD8+ T cells expressing CD43, a major ligand of CD169. We also found that the density of CD169+ macrophages had a positive correlation with the number of CD8+ cytotoxic T cells infiltrating tumor tissues. These data suggest that CD169+ macrophages in RLNs promote CD8+ T-cell-mediated antitumor immunity and are associated with a better prognosis for CRC patients. CD169+ macrophages in RLNs could be a useful marker for assessing clinical prognosis and monitoring antitumor immunity in patients with CRC.
Regional lymph nodes (RLNs) draining human malignant tumors are believed to be the first major components of the immune system to have contact with tumor cells or their products. Lymph nodes (LNs) contain various types of macrophages, such as subcapsular or medullary sinus macrophages, medullary cord macrophages, and tingible body macrophages. In particular, sinus macrophages are thought to be involved in lymph-borne tumor antigen capture and activation of antigen-specific lymphocyte responses,[2, 3] which suggests that sinus macrophages in RLNs are crucial components of the antitumor immune response in cancer patients.
CD169, also called sialoadhesin, is the foremost member of the sialic acid-binding lectin (Siglec) superfamily and is one of the extremely useful markers on sinus macrophages.[3-5] It binds sialylated glycoproteins including CD43 (sialophorin) and MUC1 and is involved in cell–cell adhesion as well as cell–pathogen interactions.[5-8] CD169 expression is found in splenic marginal metallophilic macrophages and in certain tissue macrophages in bone marrow, colon, liver, and lung, as well as in sinus macrophages.[5, 9] Monocytes/macrophages may also be induced to express CD169 under many inflammatory conditions such as systemic lupus erythematosus, systemic sclerosis, glomerulonephritis,[12, 13] atherosclerosis,[14, 15] and acute cellular rejection after transplantation of the intestine. Previous experiments with CD169-deficient mice suggested that CD169 may exacerbate an activity in experimental autoimmune encephalomyelitis through inhibition of regulatory T cell expansion. These findings suggest that CD169+ macrophages have pro-inflammatory functions and are phenotypically differentiated into classically activated (M1) macrophages.
On the basis of these findings, we hypothesized that CD169+ RLN macrophages may be involved in the development of antitumor immunity in human malignant tumors. Previous CD169 studies were mainly carried out with rodents; only a few studies described human macrophage CD169 expression.[4, 5, 9] To examine the role of CD169 in human antitumor immunity, we investigated the correlation between antitumor immunity and CD169+ RLN macrophages by using pathological specimens obtained from patients with colorectal carcinoma (CRC).
Materials and Methods
Peripheral blood mononuclear cells were obtained from healthy volunteer donors, who gave written informed consent for participation in this study. The cells were suspended in RPMI-1640 medium (Wako, Tokyo, Japan) supplemented with 10% FBS and penicillin–streptomycin (Wako), seeded in polystyrene culture plates, and incubated for 2 h at 37°C. Non-adherent cells were removed by gentle washing with PBS. The remaining monocytes were cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) (5 ng/mL; Wako) for 5 days to induce immature macrophages.
Cell ELISA assay
We evaluated expression of CD169 in human macrophages with the aid of a cell ELISA, as described previously.[18, 19] Monocytes were cultured in a 96-well plate (5 × 105 cells per well) with 5 ng/mL GM-CSF for 5 days to induce macrophage differentiation. These monocyte-derived macrophages were stimulated with the optimal concentration of: interferon (IFN)-α (50 ng/mL), IFN-β (20 ng/mL), IFN-γ (20 ng/mL), interleukin (IL)-4 (20 ng/mL), IL-6 (20 ng/mL), or IL-10 (20 ng/mL) (all from PeproTech, Rocky Hill, NJ, USA); LPS (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA); or PBS (control) for 24 h.[13, 15, 19] After cells were treated with an anti-CD169 antibody (clone HSn 7D2; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or isotype-matched control antibody (RM-4),[18, 19] samples were incubated with HRP-labeled goat anti-mouse antibody (Nichirei, Tokyo, Japan). Plates were read by using a microplate reader at 450 nm to detect signals.
Western blot analysis
We used Western blotting to determine the amount of CD169 protein. Human monocyte-derived macrophages were solubilized with Triton X-100, and the protein concentration was determined by using BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). The protein (20 μg) was run on 10% SDS-polyacrylamide gel and transferred to a PVDF transfer membrane (Millipore, Bedford, MA, USA). The membranes were incubated with anti-CD169 or anti-β-actin (Santa Cruz Biotechnology) antibodies with gentle agitation for 18 h at 4°C and were then visualized by using HRP-conjugated anti-mouse IgG antibody with ECL Western Blotting Detection Reagent (GE Healthcare, Chalfont St Giles, Buckinghamshire, UK). Signals were detected with an LAS-4000 image analyzer (Fujifilm, Tokyo, Japan).
In this study, we evaluated tumor and RLN specimens resected from 83 patients with CRC (43 men, 35 women; median age, 64 years, range: 29–90 years) and colon specimens resected from three patients with infectious colitis complicated by diverticulitis who had undergone operations at Kumamoto University Hospital (Kumamoto, Japan) from 2000 to 2006. All patients gave informed written consent for participation in the study, in accordance with protocols approved by the Kumamoto University Hospital Review Board.
Immunostaining and triple immunostaining
Tissue samples from CRC or RLNs were routinely fixed in 10% neutral buffered formalin and were embedded in paraffin. Monocyte-derived cultured macrophages were fixed with 2% paraformaldehyde on 8-well chamber slides (Sigma-Aldrich). Antigen retrieval was carried out as follows: sections were immersed in EDTA solution (pH 8.0), and samples were heated with a microwave for staining of CD169 and CD43 (clone DF-T1; Dako, Glostrup, Denmark) or with a pressure cooker for staining of CD68 (clone PG-M1; Dako) and CD8 (clone C8/144B; Nichirei). An isotype-matched mouse IgG (Dako) was used as a negative control. After reaction of the primary antibodies, samples were incubated with HRP-labeled goat anti-mouse antibody (Nichirei). Immunoreactions were visualized by using the diaminobenzidine substrate system (Nichirei). The numbers of CD68+ and CD169+ cells per mm2 in sinus areas in RLNs without metastatic cancer cells and CD8+ cells per mm2 in intratumoral areas were counted in high power fields (0.028 mm2 per field) by two independent pathologists (K.O. and Y.K.) who were blinded to information about the samples. The count data assessed by K.O. or Y.K. were averaged as described previously.
Triple immunostaining was carried out as described previously. An alkaline phosphatase-conjugated antibody (Nichirei) was used as the secondary antibody, and the reaction was visualized by using Fast Red TR Salt (for CD43 staining) or Fast Blue BB Salt (for CD8 staining) (both from Sigma-Aldrich).
Statistical analysis was carried out with JMP 10 (SAS Institute, Chicago, IL, USA). All data from in vitro studies represent the results of two or three independent experiments. Data were expressed as means ± SD. The Mann–Whitney U-test was used for two-group comparisons. Associations between different categorical variables were assessed by using anova. The cumulative survival rate was compared between two groups using the log–rank test and generalized Wilcoxon test. The simultaneous relationship between multiple prognostic factors for survival was assessed with the Cox proportional hazards model with stepwise backward reduction. Multivariate analysis included adjustment for age, tumor size, clinical stage, lymphatic invasion, LN metastasis, the ratio of CD169+ cells to CD68+ cells in RLNs and the number of CD8+ cells in tumor tissue. A P-value of <0.05 was considered statistically significant.
CD169 expression was high in IFN-stimulated cultured macrophages
We examined CD169 expression in human macrophages under various conditions. Cell ELISA revealed elevated CD169 expression in M1 macrophages stimulated with IFN or LPS; in particular, type 1 IFN (IFN-α and IFN-β)-stimulated macrophages showed strong CD169 expression (Fig. 1a). The increased CD169 expression after IFN treatment was also detected by immunoblot analysis and immunocytochemistry (Fig. 1b,c). This CD169 increase, however, was not detected in IL-4-, IL-6-, or IL-10-treated macrophages (Fig. 1a,b).
We next evaluated CD169+ cell distribution in colon tissue by means of immunohistochemistry. Many macrophages infiltrating infectious colitis specimens were positive for CD169 as well as CD68. In contrast, CD68+ macrophages infiltrating tumor tissue were completely negative for CD169 (Fig. 1d).
Number of CD169+ sinus macrophages in RLNs decreased in CRC patients with LN metastasis
We next used immunohistochemistry to investigate the expression of CD169 and CD68 in samples of RLNs, obtained from CRC patients, that contained no metastatic carcinoma cells. The number of CD169+ sinus macrophages varied for each case, although they were almost positive for the pan-macrophage marker CD68 (Fig. 2).
We then analyzed the correlation between clinicopathological features and the number of CD169+ sinus macrophages in 83 patients with CRC. We classified two groups as CD169-low cases or CD169-high cases based on the median and compared clinical factors in each group. The number of CD169+ cells per mm2 and the ratio of CD169+ cells to CD68+ cells were not associated with age, sex, clinical stage, lymphatic invasion, or vascular invasion (Table 1). In contrast, the low density of CD169+ cells was significantly correlated with cases with tumor enlargement, advanced T stage, or LN metastasis (Table 1). The number of CD68+ sinus macrophages, however, was not associated with these clinical factors.
Table 1. Clinicopathological features and the number of macrophages in regional lymph nodes (RLNs) from 83 patients with colorectal carcinoma
High density of CD169+ sinus macrophages associated with good prognosis in CRC patients
The number of CD169+ cells per mm2 and the ratio of CD169+ macrophages to CD68+ sinus macrophages were significantly correlated with a favorable overall survival in a univariate analysis (P = 0.0092 and P = 0.0012, respectively, log–rank test) (Fig. 3a,b, Table 2). Multivariate analysis indicated that the ratio of CD169+ cells to CD68+ cells was an independent prognostic factor (Table 2). No correlation existed between the number of CD68+ RLN macrophages and overall survival (Fig. 3c). As expected, LN metastasis in CRC patients was associated with a poor prognosis, as shown in previous studies (Fig. 3d). The number of CD169+ macrophages was significantly correlated with overall survival in cases with no LN metastasis (n = 55; P = 0.0236, log–rank test), but this correlation was not observed in LN metastasis-positive cases (n = 28; P = 0.1836, log–rank test) (Fig. 3e,f). The clinical stage (<stage III), tumor size <50 mm, no lymphatic invasion, and no LN metastasis were also associated with longer overall survival in the univariate analysis (Table 2). Multivariate analysis showed a significant correlation between age, tumor size, lymphatic invasion, or clinical stage and overall survival.
Table 2. Univariate and multivariate Cox regression analyses of potential prognostic factors for overall survival in patients with colorectal carcinoma (n = 83)
Density of CD169+ sinus macrophages correlated positively with CD8+ T cell infiltration in tumor tissue
Because M1 macrophage activation of the CD8+ T-cell-mediated immune response is generally accepted, we speculated that CD169+ sinus macrophages interacted with CD8+ T cells and induced T-cell-mediated antitumor immunity. To investigate the details of interactions between macrophages and T cells in vivo, we carried out triple immunostaining with mAbs for CD169, CD8, and CD43, which is a CD169 ligand. Immunostaining showed that many CD169+ cells had direct contact with CD8+ CD43+ double-positive T cells in the sinus area of RLNs (Fig. 4a).
We next analyzed the association between CD169+ RLN macrophages and CD8+ T cells infiltrating tumor tissues from CRC patients. The number of CD8+ T cells in tumor nests and tumor stroma increased significantly when many CD169+ RLN sinus macrophages were present (Fig. 2). Statistical analysis showed that the number of CD169+ cells and the ratio of CD169+ cells to CD68+ cells correlated positively with the number of CD8+ cells in tumor nest and tumor stroma (Table 1, Fig. 4b). The CRC patients with abundant CD8+ T cell infiltration also had a favorable overall survival (Fig. 4c, Table 2).
Although a number of previous studies of CD169+ macrophages led to advances in understanding their distribution among various macrophages in rodents, only a few described regulation of CD169 expression in human macrophages.[4, 5, 9] In the present study, we showed, by means of in vitro experiments, high expression of CD169 in IFN-stimulated M1-type human macrophages, and we suggest that CD169 expression may be seen in exudative macrophages in human tissues. We observed CD169 expression in LN sinus macrophages and resident macrophages in the submucosa of the colon; however, our preliminary observations also showed expression of CD163, which is a specific marker of alternatively activated (M2) macrophages,[18-20] in these cells. Therefore, we believe that CD169 is not a marker restricted to M1 macrophages but is rather a marker of IFN-induced inflammatory macrophages. That Th1-type lymphocytes secrete IFNs is well known, so CD169 expression may be associated with inflammatory conditions in the sinus areas of LNs.
We also showed that the higher density of CD169+ RLN sinus macrophages was correlated with increased numbers of cytotoxic CD8+ T cells infiltrating tumor tissue and with a favorable overall survival of CRC patients. Because our present study and many previous studies[23-26] indicated a relationship between infiltrating CD8+ T cells and clinical prognosis in CRC, we suggest that CD169+ RLN macrophages are closely related to activation of CD8+ T-cell-mediated antitumor immunity. Because the close association between infiltrating CD8+ T cells and clinical prognosis was reported not only for CRC but also for other malignant tumors, including endometrial carcinoma, urothelial carcinoma, gastric cancer, breast cancer, and melanoma,[27-33] similar mechanisms related to CD169+ cells and T cells may occur in RLNs.
Recently, Asano and colleagues found that CD169+ LN macrophages engulfed tumor cell antigens and caused the proliferation of antigen-specific cytotoxic T cells in a tumor transplantation mouse model. Barral and colleagues showed that CD169+ macrophages acted as antigen-presenting cells that control invariant natural killer T cell activation in murine LNs. Xiong and colleagues showed that increased CD169 expression on monocytes was related to the CD4+ or CD8+ T cell proliferation, and CD169 knockdown by siRNA could attenuate T cell proliferation. Our study showed that CD169+ macrophages had direct contact with CD8+ CD43+ T cells in sinus areas of RLNs, and this finding indicated that CD169–CD43 ligation in the RLN mediated cell–cell interaction between sinus macrophages and CD8+ T cells. Although the detailed mechanism behind CD169–CD43 ligation and consequent CD8+ T cell activation is unclear, these data suggested that CD169+ macrophages played an important role in antitumor immunity in patients with CRC by presenting tumor antigens to cytotoxic lymphocytes. To address this problem, we are in the process of investigating CD169-mediated CD8+ T cell proliferation by means of in vitro experiments.
In conclusion, we showed the significance of CD169+ sinus macrophages in RLNs obtained from patients with CRC. A higher number of CD169+ macrophages and an increased ratio of CD169+ macrophages to CD68+ macrophages in RLNs were significantly associated with better clinical prognosis; moreover, the ratio of CD169+ macrophages to CD68+ macrophages was an independent risk factor for overall survival. Because the ratio of CD169+ macrophages to CD68+ macrophages was also related to the number of CD8+ T cells infiltrating tumor tissues, we believe that CD169+ macrophages are involved in activation of cytotoxic T cells in RLNs (Fig. S1). Evaluation of the CD169 antigen in RLNs may aid estimation of clinical prognosis and monitoring of the antitumor immune response in patients with CRC.
We sincerely thank Ms Yui Hayashida, Mr Osamu Nakamura, and Mr Takenobu Nakagawa for their valuable technical assistance. This study was supported in part by Grants-in-Aid for Scientific Research (B23790407, B24790353) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.