[The effect of sera of breast cancer patients on NK cell activity].

Introduction: The immune system may influence tumorogenesis through natural killer (NK) and specific T cytotoxic cells. It is considered that NK cells, which constitute 15% of peripheral blood lymphocytes, have a main role in defense, due to their ability to destroy tumor cells in situ or in the blood stream in first contact without major histocompatibility restriction. Determination of NK cell activity in malignancies indicates that the evaluation of their activity is more reliable than their number, as well as that IL-2 and interferons increase, while prostaglandines and some hormones decrease their activity. With regard to this, investigations of the effect of sera of patients with malignancies on NK cell activity are being performed [10] in order to see whether the impairment of this activity, which is common in advanced disease, may be due to the presence of different factors in their sera or is the consequence of a defect in NK cells, themselves. This work analysis the influence of sera of healthy persons and patients with different stages of breast cancer on the activity of NK cells of controls and patients with malignancies.

Materials And Methods: NK cell activity was evaluated in 58 patients with stage I-III, 11 patients with stage IV of breast cancer and 18 healthy controls. The sera of 30 patients with stages I-III (CaSa), 11 patients with stage IV of breast cancer without or with metastases (CaSb, CaSm, respectively) and 35 healthy controls (ZS/HS) was used for analysis of their effect on NK cell activity. Foetal calf sera (FCS) were used as controls. Isolation and cultivation of peripheral blood lymphocytes: Lymphocytes from 30 ml of blood were isolated by sedimentation on lymphoprep gradient (Nycomed, Norway) and resuspended in RPMI 1640 culture medium without sera in concentration of 4 x 10(6) cells/ml of medium Lymphocytes of each person were treated in a short term culture for 18 h, simultaneously with 10% of inactivated FCS, 10% inactivated healthy sera (HS) and 10% inactivated sera of patients with different stages of breast cancer (CaSa, CaSb, CaSm). NK cell assay: NK cell activity was evaluated as previosly reported by the radioactive 51-Cr release assay. Namely, 100 microl of lymphocytes as effector cells (E) in concentration of 4 x 106 cells/ml of medium and three 1:1 dilutions there of were mixed with 100 ul of target tumor cell line K 562 (T) previosly labeled with 51-chromium (As = 100 microCl) concentration of 5 x 10(5) cells/ml of medium in 96 round bottom plates and incubated for 4 h in an incubator at 37 degrees C with 5% CO2 and humidity. After that the plates were centrifuged and 100 microl of supernatants were used for determination of 51-Cr release in a gamma-counter and expressed in counts per minute (cpm). The percent of specific NK cytotoxicity was determined by the following formula: ((cpm in experimental release - cpm in spontaneous release/ (cpm in maximal release - cpm in spontaneous release)) x 100 where the spontaneous and maximal release were obtained by incubation of tumor cells in culture medium i.e. with 5% Triton x 100 solution, respectively. The results were interpreted by the Mann-Whitney statistical method. RESULTS NK cell activity determined in 18 healthy controls was x = 57.75 +/- 2.21% (E:C=80:1, in patients with breast cancer in clinical stages I-III n=58) it was x = 36.64 +/- 1.92% (E:C=80:1) which is significantly (p < 0.01) below that found in controls, while in patients in stage IV (n=11) it was x = 18.31 +/- 1.38% (E:C=80:1) which is significantly (p < 0.01) below the stages I-III (Table 1). Short term in vitro treatment of PBL in culture medium with FCS gave a significant increase in NK cell activity of both groups of patients: in stages I-III and stage IV (Table 1). Treatment of PBL of patients with stages I-III with sera CaSa, CaSb and CaSm gave a progressively greater inhibition of their NK cell activity compared with FCS (Figure 1). The same type of treatment of PBL of patients with stage IV of breast cancer with HS and CaSb also showed a significant inhibition of their NK cell activity compared to treatment with FCS (Figure 2). Treatment of PBL of healthy controls with CaSa, HS and CaSm demonstrated the same type of progressive inhibition of NK activity (Figure 3).

Discussion: In this work it has been shown that patients with breast cancer in clinical stages I-II, and especially stage IV, have a significantly decreased NK cell activity compared to controls. However, the treatment of both controls' and patients' PBL with sera of patients with different stages of this disease changed NK cell activity. The sera of the first three stages of breast cancer increased this activity, while sera of advanced disease caused a progressive decrease compared to control treatments with FCS. Some of these results were partially obtained in different investigations indicating the presence of stimulative factors in sera of patients with locoregional disease, and inhibitory factors in sera of advanced disease, as well as a slight, probably homeostatic, inhibitory effect of sera of healthy controls 2 These results indicate that the changes in NK cell activity are governed more by the factors in the present sera than by the changes in the activity of NK cells, themselves. The nature of these factors in sera should be further investigated.