Impaired Th1-Related Immune System in Cancer Patients: Comparable Down-Regulation In Early Stage And Advanced Stage

Katsuaki Uno¹, George Housokawa² and Yuji Ueda³

Department of Surgery, Comfort Hospital¹, Yokohama Japan, Department of Cardiology, Shonan Kamakura Sogo Hospital², Kamakura Japan, Department of Internal medicine, Comfort Hospital³, Yokohama Japan

Summary

Background

Interleukin 12 (IL-12), Interferon- y (IFN- y) and Tumor necrosis factor- ß (TNF- ß are known to induce proliferation and differentiation of Th1 cells. These "Th1-related" cytokines are shown to negatively modulate the growth of tumors. In animal models, Th1 cells are shown to be activated shortly after the implantation of tumor cells and exert antitumor effects, but suppressed and overwhelmed by expanding Th2 cells as tumor cells grow. In this study, we ask whether Th1 system including these cytokines is actually modulated in cancer patients.

Methods

Patients diagnosed cancer of various organs, and 100 control subjects without cancer were enrolled to the study. The production of Th1-related cytokines (IL-12, INF- y and TNF- ß) by peripheral blood mononuclear cells, and the induction of Th subsets (Th1 and Th2) were measured and compared between patients and control subjects after mitogen stimulation in vitro. Natural killer (NK) cell activity was also measured. To evaluate the influence of disease progress we selected stomach cancer and colon cancer due to their common criteria for staging. Each parameter was compared between cancer patients in early stage, in advanced stage and control subjects.

Findings

Phytohemagglutinin(PHA)-induced IL-12, IFN- y and TNF- ß in cancer were significantly lower than those in control. Th1 cell induction was also lower in cancer than that in control we found no significant difference in Th2 induction between cancer and control. NK cell activity was not up- or down-regulated in cancer patients. When patients with either stomach cancer or colon cancer were grouped to two stages based on the common criteria for staging, suppressed cytokines and Th1 cells were seen in the early stage as well as the advanced stage. None of these parameters differed between the two stages of cancer. Collectively, results show that suppressed Th1-related immunological parameters already exist at early stage of cancer and persist through the disease progress.

Interpretation

Th1-related immune system appears already impaired in early stage of cancer which suggests either; (a) tumor cells even in small size have significant effects on immune system and have already down-regulated Th1-related function or; (b) defective Th1 system precedes the emergence of tumor cells. Our data may favor that the latter is the case. Our results also suggest that Th1- related immune parameters would be useful immunological markers for cancer screening.

Introduction

Although cancer therapies have progressed it is still not easy to find the disease in its early stage. Because the incidence of cancer is still increasing globally periodical health check is getting more important and preventing medicine need to be highlighted. But unfortunately, conventional methods including hematological, biochemical, urinal examinations as well as X-rays are not sensitive enough to detect cancer in the early stage. In this concern, the development of better techniques to detect cancer in the early stage.

The acquired immunity is characterized by the function that immune competent cells including T cell and B cells recognized foreign antigen with antigen specific manner. From animal experiments the antigen specific mechanism1 was shown to be activated and functioning in the cancer immunity whereas antigen non-specific mechanism2 is also functioning. The finding of Th1 and Th2 subsets based on the cytokine production pattern gave us a tremendous help in understanding immune system. Studies have revealed that IFN- y, and TNF- ß produced by Th1 cells and IL-12 by APC through the cross talk between these cells are key factors for the differentiation of Th cells to Th1 cells which are responsible for cancer immunity. Together, these cytokines seem to modulate and optimize the immune responses including antitumor responses originally initiated with antigen specific or non-specific manner.

In this study, we shed light on Th1-related cytokines and Th subsets in cancer patients and asked whether these parameters are actually modulated in cancer. To the aim mitogen-induced cytokines and Th subsets were measured in cancer patients and compared with those in control subjects. Then we selected stomach cancer and colon cancer for the evaluation of the influence of disease progress on immunological parameters. We found impaired Th1 - related immunological parameters not only in advanced stage but also in early stage of cancer, suggesting that defective anti-tumor immunity already exists in early stage of cancer. We propose that Th1-related immunological parameters may be useful markers for cancer detection at early stage.

Methods

Patients

Patients who had been diagnosed cancer, aged 16 - 87 years, at various organs were enrolled (Table 1). Patients who had experienced therapies including surgical operation, chemotherapy and radiation were not included. When we selected patients with stomach cancer or colon cancer for the evaluation of the influence of disease progress on immunological parameters. They were grouped to the early stage, cancer cells stay within mucosa (m) or submucosa (sm), or the advanced stage; cancer cells invade beyond the submucosa (Table 2). 100 individuals without cancer, aged 27 - 72, were enrolled as controls. Consent was obtained from all the patients and control subjects.

Study Design

For cytokine assay Heparinized peripheral blood cells were diluted by phosphate buffered saline without calcium and magnesium (PBS (-)). Centrifuged on Ficoll-Conray at x400 G for 20 min, mononuclear cells were collected. After washing, mononuclear cells were resuspended in RPMI-1640 (IBL, Co., Fugioka, Gunma, Japan) with 10% FCS (Cancera, Rexdale, Ont.) with the concentration of 1 x 106 cells/ml of lymphocytes. Lymphoctes (2x10/200 µ 1) and 20 µ g/ml of phytohemagglutinin P (PHA-p; Difco, Laboratories, Detroit, MI) were added to 96 holes microtiter plates, with 220 µ 1 in final volume. After incubation at 37ºC for 24 hours with 5% CO₂ supernatants were collected for cytokine assay. IL-12 was measured by enzyme linked immuno solvent assay (ELISA) kit (R&D Systems, Minneapolis, MN). IFN- y and TNF- ß were measured by ELISA kits (IFN- y; Biosource, Nivelles, Belgium, TNF- ß; JIMRO Gunma, Japan).

All cytokines were measured with the way as manufacturers recommended.

For identification of Th cell subsets Heparinized peripheral blood were stimulated with 25 ng/ml of PHorbol 12-Myristate 13 Acetate (PMA, Sigma Chemical Co., St. Louis, MO), 1 µ g/ml of ionomycin (Sigma Chemical Co., St. Louis, MO) and 10 µ of Brefeldin-A (Sigma Chemical co., St. Louis, MO) at 37ºC with 5% CO₂ for 4 hours. 300 µ 1 of stimulated blood was added by 20 µ 1 of peridinin chlorophyll protein (percept)-conjugated anti-CD4 antibody (Immunotech, Marseille, France) incubated at room temperature for 15 min. 4 µ 1 of x 10 Lysing Solution (BDIS, San Jose, CA) was added and incubated for fixing cell surface epitopes, lysing red blood cells and pretreatment for permialization. Then 1.5 µ 1 of x 10 Lysing Solution (BDIS, San Jose, CA) was added and incubated at room temperature and for 10 min. For staining of intracellular cytokins 20 µ 1 of fluorescein isothiocyanate (FITC)-conjugated anti-IFN- y antibody (BDIS, San Jose, CA) and phycoerythrin-conjugated anti-IL-4 antibody (BDIS, San Jose, CA) were added and incubated for 30 min. at room temperature. FITC-conjugated IgG2a/PE-conjugated IgG1 antibodies (BDIIS, San Jose, CA) were used as negative controls. The FACS analysis was done with FACScan (BDIS, San Jose, CA).

For the assay of NK cell activity target cells (K-652) 3x106 were incubated with 100 µ Ci of 51Cr-Sodium Chromate (Diichi Radiosotope, Tokyo, Japan) at 37oC for 1 hour. After washing 0.1 x 105/10 µ 1 of target cells were mixed with 1x106/200 µ 1 of effector cells. After incubation at 37ºC for 3.5 hour supernatant was collected and radioactivity was measured NK cell activity was calculated with following formula. NK cell activity (% lysis) = (Test cpm- spontaneous cpm/Total cpm - spontaneous cpm)

Statistical Analysis

The analyses were performed with a commercially available statistical package (SPSS Ver 9.0). Because all cytokines, Th subsets and NK activities are not normally distributed we used Mann-Whitney U test for the comparison of the two distributions and Kruskal-Wallis test with Boneferroni test for the three distributions. The results are presented using median values with 25th and 75th percentiles.

Results

Patients (206 men, 215 women; age range 16 - 87 years [median 57]) and 100 controls were enrolled (61 men, 39 women, age range 27 - 72 years [median 53]). PHA-induced median IL-12 was 7.8 pg/ml in the patients and 28.5 pg/ml in the control group (p<0.001, Mann Whitney U test, Fig. 1). The median INF- y and TNF- ß were 6.8 IU/ml and 1420 pg/ml in the whole patients and lower than 29.9 IU/ml and 2450 pg/ml in the control respectively (INF- y; p<0.001, TNF- ß; p<0.001, Mann Whitney U test). The median Th1 subset was 22.1% in the patients and 27.5% in the control (p<0.001, Mann Whitney U test). The median Th2 subset was 2.6% in whole patients and had no difference to 2.4% in control (p=0.21 Mann Whitney U test). NK cell activity was 42.5% in cancer and higher than 34.5% in control (p<0.001, Mann Whitney U test).

To elucidate the influence of disease progress to the immune parameters, we selected patients with stomach cancer or colon and categorized them to two groups; early stage and advanced stage. When cytokines were compared between the early stage and control, all of three cytokines (IL-12; 7.8 pg/ml, INF- y; 6.1 IU/ml, TNF- ß; 1305 pg/ml) in the early stage were lower than those (IL-12; 28.5 pg/ml, INF-y; 29.9 IU/ml, TNF- ß; 2450 pg/ml) in control respectively (IL-12; p<0.001, INF- y; p< 0.001, TNF- ß; p<0.001, Krukal Wallis and Bonferroni test). On the other hand, there was no significant difference between cytokins (IL-12; 7.8 pg/ml, INF- y; 6.1 IU/ml, TNF- ß; 1340 pg/ml) in the advanced stage respectively (IL-12; p=1.000, INF- y; p=1.000, Kruskal Wallis and Bonferroni test)(Fig. 2). The median of Th1 subset in the early stage was 19.0% and lower than 27.5% in the control (p<0.001, Krukal Wallis and Bonferroni test) (Fig. 2). The median Th1 subset (19.0%) in the early stage was not different from that (23.5%) in the advanced stage (p=0.34, Kruskal Wallis and Bonferroni test) (Fig. 2). Th2 subset was not significantly different between three groups (data not shown) (Fig. 2). NK cell activity was not significantly different between three groups either (data not shown) (Fig. 2).

Discussion

Our results demonstrate that: (a) Mitogen-induced Th1-related cytokines (IL-12, IFN- y; TNF- ß) were lower in patients with cancer than those in controls respectively; (b) Th1 subset induced in cancer was lower than that in control, whereas there was no difference of Th2 subset between cancer and control; (c) NK cells activity was up-regulated in cancer compared to that in control; (d) Th1-related cytokines in early stage of cancer were down-regulated and comparable to those in advanced stage; (e) Th1 Induction in early stage was down-regulated to the comparable level in advanced stage; (f) No difference was found in NK cell activity when patients were grouped to the two stages.

IL-12 is originally found as a cytokine that stimulates natural killer cells.³ Studies revealed that IL-12 induces IFN- y production by NK cells4 and T cells5, stimulates the proliferation of T cells5 and NK cells3 and augments NK cell-mediated cytoxicity6, 7 and cytolytic T cell responses^{8, 9, 10.} Since the finding of Th1 and Th2 subsets11 the mechanism of positive interaction between IL-12 and IFN- y for Th1 induction has been defined.^{4, 12-14} Activated antigen presenting cells (APCs) produce IL-12, which stimulates Th1 cells to proliferate and differentiate to effector cells including cytoxic T cells and delayed type hypersensitivity (DTH)-committed T cells. In the presence of IL-12 Th1 cells produce IFN- y, hypersensitivity (DTH)-committed T cells. In the presence of IL-12 Th1 cells produce IFN- y, with which the Th1 cell itself proliferates and, in turn, IFN- y augments IL-12 production by APCs. On the other hand Th2 cells, which are mainly involved in allergic reaction and Ig production, are dependent on APC-derived IL-1, instead of IL-12, and produce IL-4 with which Th2 cells proliferate. Th1 cells are negatively regulated with IL-1015, 16 and IL-4 produced by Th2 cells with the mechanism of blocking macrophage-derived IL-12 production.¹⁵ Thus Th1 and Th2 negatively regulates each other and are mutually inhibitory.

Which subset of Th cells to be activated is dependent on multiple factors. For example, in BALB/C mice, infected with Leishmania, Th2 cells are activated and lead to Ig production, especially IgE and the mouse cannot deplete the organism. In contrast, in C57/BL6 mice Th1 cells are activated with Leishmania and exert cell-mediate immunity16 leading to the depletion of the organism. This demonstrates that same antigens are to activate differential Th subsets if provided by particular generic background.¹ Other studies show that a peptide can activate either Th1 or Th2 cells in the same mice depending on the provided accessory signals, ^{18, 19} which means antigen specific immunological response can be modulated to either Th1 dominant or Th2 dominant.^{13, 20, 21} In other words, the decision to which subset Th cells differentiate is modulated by accessory signals including cytokines.

In cancer immunity responsive subset is again Th1 cells. For example, in a mouse model, shortly after implantation of tumor cells Th1 cells were activated to show antitumor responses in the presence of Th1-related cytokines. But later as tumor increases in size, Th1 system is down regulated and overwhelmed by Th2 system, which allows tumor cells to grow.²⁵ Therefore, IFN- y and IL-1222-24 also seem to participate in T cell-mediated antitumor immunity. One question here is whether the anti-tumor activity can be initiated or mediated by antigen specific manner. Ogata et al. showed that C3H/He mice initially bore a MCH-1-A1 tumor and received surgical resection of the tumor rejected a second challenge of same tumor cells3. This illustrates that once tumor cells are successfully removed even with the way other than immune mechanism the antitumor immunity can be established. In addition they showed C57BL/6 x C3H/He mice successfully rejected OV-HM tumor with i.p. injection of IL-12, otherwise the tumor will outgrow. Of importance is provided by findings that the mouse initially bearing the OV-HM tumor and treated with IL-12 only rejects a second challenge of the same OV-HM tumor but not unrelated MCH-1-A1 tumor cells. In similar experiments the anti-tumor activity of IL-12 was completely abrogated with administration of anti-IFN- y mAb before IL-12 treatment. These results demonstrate that IL-12 and IFN-y are actually enhancing cancer immunity which is mediated, at least in part, by "antigen specific mechanisms. On the other hand, Siders et al demonstrated that a recombinant adenovirus encoding IL-12 can inhibit hepatic metastasis of murine tumors even after the depletion of T cells in normal mice or even after the depletion of NK cells in SCID mice2, which suggests that IL-12 also evokes its antitumor activity initiated with antigen non-specific manner.

In this study we showed that the induction of IL-12 and IFN- y as well as TNF- ß in whole patients with cancer were lower than those in controls, which appeared compatible with above findings in mice. When patients with stomach cancer or colon cancer were grouped to the early stage and advanced stage, we expected that significantly different amounts of cytokines could be observed between the two stages. But surprisingly, these cytokines are already down regulated at the early stage and we found no difference between early stage and advanced stage. This provides us two possible cases; (a) even at clinically early stage of the cancer immune system is already impaired due to the presence of cancer and it is actually advanced stage immunologically; (b) immunological impairment precedes the emergence of cancer. If the latter is the case, it should be taken into account that cytokine abnormality itself may be involved in the etiology of cancer by down-regulating the anti-cancer defense mechanism. In this study we employed in vitro mitogenic stimulation to evaluate cytokine production and Th subset induction using mitogenic stimulation. Obviously in this system we are determining the function of not only tumor specific T cell clone(s) but also other T cells. Therefore, the fact that we could detect abnormal Th1 function in this system suggests that detected abnormalities are not limited to tumor specific Th1 subset and its precursor but also other T cell repertoires that would be committed to other specificity. This idea may favor the latter interpretation mentioned above.

In the present study NK cell function was not impaired in cancer at any stage and did not appear to be related to cancer susceptibility though it was shown, in a study, that NK cells were required for IL-12 induced antitumor response in mice. ²⁷

Taken together, our data strongly suggest that, even in early stages of cancer, the Th1 system is already impaired and this is quite different from the condition of the animal model, where the Th1 system is activated shortly after the implantation of the tumor. Also, we should pay more attention to the possible role of immunological conditions for the emergence of cancer. It should be interesting to measure these parameters before and after the surgical resection of early cancer. Our results also provide chances to detect cancer at an early stage or even earlier than clinical onset by means of immunological parameters.

Finally, it is well accepted that multiple factors including gene level, environmental, nutritional, hormonal, etc. are involved in oncogenesis. But these factors may be too prevalent to stay the incident of cancer to the level actually observed. We think that preventive mechanism by immune system is a key factor to determine whether cancer cells can survive or will be deleted.

Table 1:

	Cancer Patients (n=421)	Controls (n=100)
Demography
Sex (male/female)	206/215	61/39
Median age (years)	57	53
Age range (years)	16-87	27-72
Organ (male/female)
Thyroid gland	1/6
Lung	51/30
Esophagus	16/2
Stomach	38/43
Colon	29/27
Rectum	23/7
Liver	12/10
Gall bladder	3/1
Bile duct	3/2
Pancreas	10/8
Kidney	3/1
Ureter	0/1
Bladder	6/0
Prostate	10/0
Uterus	0/20
Ovary	0/21
Breast	0/37

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