Prostaglandin sensitivity of the PHA-response of blood lymphocytes following radiation therapy for breast cancer

The reduction of mitogen responses of blood lymphocytes which occur after radiation therapy for breast cancer, known to be largely due to inhibitory monocytes, can partly be reverted by indomethacin which is an inhibitor of prostaglandin (PG) synthesis. This may indicate that PG-synthesis by blood monocytes is increased after irradiation or that PG-sensitivity of the lymphocyte population is increased. To test the latter possibility the sensitivity of the PHA-response of blood lymphocytes to varying concentrations of PGE2 and PGD2 was examined in 15 patients with breast cancer before and up to 6 months after local radiation therapy (46 Gy). The results showed that the sensitivity of the PHA-response was not significantly changed after treatment suggesting that the immunosuppression observed after irradiation is partly due to an increased production of PGs rather than an increased PG-sensitivity of lymphocytes.     

Relationship between T lymphocyte levels and lymphoproliferative responses to mitogens and alloantigens in lung and breast cancer patients.

Lung and breast cancer patients were studied for relationships between lymphocyte function, as measured by lymphoproliferative (LP)³ assays, and levels of circulating thymus-derived lymphocytes (T lymphocytes) measured by rosette formation with sheep erythrocytes (E rosettes). LP responses to the T lymphocyte mitogens phytohemagglutinin (PHA) and concanavalin-A (Con-A) and to alloan-tigens in one-way mixed leukocyte cultures (MLC) were assessed in a microculture assay. Total T lymphocyte levels were determined using an E rosette assay performed at 4°C and a subpopulation of T lymphocytes with high affinity receptors for E rosettes was measured in a 29°C assay. Using the relative proliferation index (RPI) which compares patient reactivity to normal reactivity, we found that one-third to one-half of the lung and breast cancer patients had depressed LP responses to at least one of the stimulants. In contrast, only 21% of the breast and 25% of the lung cancer patients had low levels of total E-rosette-forming cells (E-RFC) as measured by the 4°C E rosette assay. Using the 29°C E rosette assay, we found that a significantly higher proportion of the patients demonstrated decreased levels of this subpopulation of lymphocytes and in both patient groups the depression noted in functional assays was closely paralleled by the percentage of patients with abnormal levels of high-affinity T lymphocytes. To further examine this apparent relationship between high-affinity E-RFC levels and LP results, the correlation between these measures in individual patients was examined. Regression analysis indicated a significant correlation between mitogen and alloantigen responses in both patient groups as well as between total T lymphocyte and high-affinity T lymphocyte levels. However, there was no significant correlation between LP responses and T lymphocyte levels. Studies in which MLC and PHA reactivities, 4°C and 29°C E rosette levels were determined on the same peripheral blood leukocyte (PBL) sample demonstrated that a number of lung and breast cancer patients with abnormal LP responses had normal total and high-affinity T lymphocyte levels. Patients with a decrease in high-affinity E-RFC but normal levels of total T lymphocytes had a high incidence of depression to PHA (56 and 82% in breast and lung cancer patients respectively) but fewer had depressed MLC reactivity (33 and 71% in breast and lung cancer patients, respectively). In patients with low levels of total and high-affinity T lymphocytes, there was a high incidence of depression in responses to both PHA and MLC. The results of this study indicate that some cancer patients may have depressed LP responses in association with depressed levels of T lymphocytes, but that other patients may have depressed functional reactivity despite normal levels of E-RFC.     

Cellular immune reaction to human malignant melanoma and breast carcinoma cells.

An in vitro microassay was used to study cytotoxic reactivity of lymphocytes of 19 patients with malignant melanoma, 8 patients with breast carcinoma, and 18 normal subjects on cell cultures of malignant melanoma and breast carcinoma. In each of the twelve experiments, peripheral blood lymphocytes from individuals with and without cancer were tested simultaneously on two or three different target cells. Cytotoxic reactivity, evaluated by a comparison of the number of target cells remaining after incubation with lymphocytes with those incubated with medium only, was found in 20 cancer patients (74%) and 13 individuals without cancer (72%). The strength of lymphocyte reactivity of the cancer and of the non-cancer group did not differ significantly. Of the 27 cancer patients, 8 were positive only on the homologous target cells, 7 only on the opposite cells, and 5 on both types; 7 were negative. Short-term melanoma cell cultures were more lysable than were established cell lines; however, no direct correlation between the growth rate during the test period and susceptibility to lysis was seen. The blood group of the lymphocyte donors had no influence on cytotoxic reactivity.     

紫杉醇对早期乳腺癌患者外周血淋巴细胞的影响

背景与目的：免疫功能是影响乳腺癌预后的重要因素之一。外周血及肿瘤内浸润的杀伤性T细胞计数能够预示乳腺癌的总生存率。另外,辅助化疗亦是提高术后乳腺癌患者无复发生存率及总生存率的重要环节。选择既对肿瘤细胞具有足够的杀伤作用,又能最大限度保留患者免疫功能的化疗药物对提高乳腺癌患者生存率具有重大意义。本研究旨在比较两种化疗方案,即蒽环类药物为主的CEF(环磷酰胺、表柔比星、5-氟尿嘧啶)方案以及蒽环类药物联合紫杉醇的EC序贯P(紫杉醇)方案对早期乳腺癌患者外周血淋巴细胞的影响。方法：回顾性分析自2012年11月-2013年5月在中山市人民医院乳腺外科行CEF方案(CEF组,n=20)或者EC序贯P方案(EC-P组,n=22)的早期乳腺癌术后患者的临床病理特征以及化疗前后患者外周血淋巴细胞的变化。关注的外周血淋巴细胞指标包括：总淋巴细胞计数以及T淋巴细胞、杀伤性T细胞、辅助性T细胞、活化T细胞和自然杀伤(NK)细胞的比例。结果：EC-P组患者较高危,临床分期、肿瘤大小、腋窝淋巴结状况、雌孕激素受体表达水平、组织分型等差异均有统计学意义(P＜0.05)。化疗前,两组患者的各项外周血淋巴细胞指标差异无统计学意义。化疗过程中,CEF组患者化疗4个及5个疗程后,外周血淋巴细胞总数分别为(1 077±359)个/μL和(1 181±271)个/μL,明显较化疗前[(1 607±322)个/μL]减少,差异有统计学意义(P<0.05)。EC-P组患者化疗4个及5个疗程后,外周血淋巴细胞总数分别为(1 500±312)个/μL和(1 623±468)个/μ L,与化疗前[(1 746±576)个/μ L]比较,差异无统计学意义(P>0.05)。两组活化T细胞比例均随化疗疗程升高,CEF组化疗前(11.8±7.1)%,第5个疗程后(23±9.3)%,差异有统计学意义(P<0.05);EC-P组化疗前(11.8±5.8)%,第5个疗程后(17.6±8.2)%,差异有统计学意义(P<0.05)。在EC-P组中,序贯使用1次紫杉醇后,辅助性T细胞比例为(37.8±5.7)%,较化疗前[(41.3±4.3)%]及使用紫杉醇前[(41.9±5.6)%]均显著下降(P<0.05);NK细胞比例为(21.5±5.2)%,较化疗前[(15.3±7.6)%]及使用紫杉醇前[(14.9±5.9)%]均显著升高(P<0.01)。结论：相较于CEF方案,EC序贯P方案对术后乳腺癌患者的免疫功能影响较小。同时,紫杉醇能增加乳腺癌患者NK细胞比例,而不影响总T淋巴细胞及杀伤性T淋巴细胞比例,在保留早期乳腺癌患者抗肿瘤免疫功能方面具有优越性。     

Impaired B lymphocyte reactivity in patients after radiotherapy

The effect of therapeutic irradiation upon B lymphocyte function was investigated in patients with various malignancies. The test system used was a reverse hemolytic plaque assay, which made it possible to study the activation and differentiation of B lymphocytes into immunoglobulin-secreting cells (ISC). Peripheral blood lymphocytes from normal individuals and patients before and after radiotherapy were stimulated in vitro with the polyclonal B cell activator pokeweed mitogen, and the number of ISC was estimated. B cell reactivity was markedly reduced in those patients who had received irradiation within the last six months. In patients in whom radiotherapy had been terminated more than 12 months before the lymphocytes were tested, B cell reactivity was comparable to that of patients prior to radiotherapy. By means of marker analyses, there was a reduction of B lymphocytes and T lymphocytes in the peripheral blood with a preponderance of T helper cells. Several mechanisms--e.g., reduced or defective B cell differentiation, altered regulatory T-helper or suppressor cell function or activation of suppressive monocytes--could be responsible for impaired B cell reactivity after radiotherapy.     

Suppressor cell activity of lymphocytes infiltrating human lung and breast tumours.

Tumour-infiltrating lymphocytes (TIL) can be isolated from human lung and breast tumours by the stepwise application of velocity and density sedimentations on discontinuous Ficoll-Triosil or bovine serum gradients. The populations obtained showed variable composition with respect to lymphocyte subsets but generally corresponded to that of peripheral blood lymphocytes (PBL) containing primarily T cells (mean, 49% E rosettes; 15% EA rosettes). Comparison of reactivity to PHA in 15 individuals revealed a significant deficit in reactivity in the TIL population compared with blood and lymph-node lymphocytes. TIL showed no natural killer (NK) activity, no response to autologous tumour in mixed lymphocyte target interaction tests (MLTI) and no cytotoxicity against autologous tumour, in agreement with previous findings. In order to investigate this depressed reactivity, we measured the effect of admixture of mitomycin-treated TIL to PBL on PHA and MLTI responsiveness in nine individuals. Eight of these nine showed reduced reactivity to PHA (mean depression of responsiveness, 49%) and five subjects who were responsive to autologous tumour, showed markedly reduced reactivity in the presence of TIL (mean reduction, 65%). In a case of medullary carcinoma of breast it was possible to revover sufficient TIL to allow fractionation on nylon wool columns. Passage through these columns resulted in a higher level of inhibition of PBL reactivity than that seen in unpassed or column-attached lymphocytes. These data show that TIL have suppressive activity which may account for their poor performance in other assays of effector and memory function.     

Radiotherapy and tumor immunity--analysis using monoclonal antibodies

In order to better understand the immunologic effects of irradiation, blood levels of lymphocyte subsets were sequentially monitored in 37 patients before and during irradiation treatment for lung cancer. Tumor infiltrating lymphocytes induced by radiotherapy were analysed by the avidin-biotin-horseradish peroxidase method. 49 cases of head and neck cancer were examined. In some cases, remarkable infiltration of lymphocytes was observed surrounding cancer cells during radiotherapy. This infiltration was mainly composed of anti-Leu-3a + 3b positive lymphocytes, and HLA-DR positive cancer cells were remarkably observed.     

Lymphocyte reactivity in patients with carcinoma of the breast and large bowel.

The reactivity of lymphocytes from patients with either carcinoma of the breast or large bowel has been studied using the human to mouse normal lymphocyte transfer (NLT) reaction. It was found that, in the case of breast cancer, there was a direct correlation between the clinical stage and a reduced NLT reaction. Only patients with regional lymph node or generalized metastases showed significantly reduced lymphocyte reactivity. However, in the case of large bowel cancer there was a generalized reduction in NLT reactivity which was independent of the clinical stage. Incubation of lymphocytes from individuals without neoplastic disease in serum or plasma from breast cancer patients, showing reduced NLT reactivity, resulted in a reduced NLT reaction. This appears to be indicative of the presence of circulating "blocking factor" in such patients.     

Changes in peripheral lymphocyte subsets during radiotherapy for lung cancer patients

In order to better understand the immunologic effects of irradiation, blood levels of lymphocyte subsets were sequentially monitored in 37 patients before and during irradiation treatment for lung cancer. During irradiation, the peripheral blood levels of each T cell subgroup, OKT3-reactive (OKT3+), OKT4+, OKT8+ and OKT11+ lymphocytes showed similar radiosensitivity. No selective depletion of either OKT4+ or OKT8+ lymphocytes was seen. The levels of OKT6+ and OKT9+ lymphocytes were different depending on the case. At the end of irradiation, the percent of lymphocytes bearing the OKT10 antigen increased significantly. As to OKM1+ and OKIa1+ lymphocytes, the levels were almost consistent but showed a rather big standard deviation.     

Application of the immunohistochemical method as a predictive assay in radiotherapy of squamous cell carcinoma of oropharynx and hypopharynx

We analyzed various subsets of lymphocytes infiltrated into cancer tissues from 15 patients (10 cases of oropharyngeal cancer and 5 of hypopharyngeal cancer) before treatment and after irradiation with approximately 10 Gy (1,000 rad) and 30 Gy by the method of biotin-avidin-horseradish peroxidase using mouse monoclonal antibodies. The infiltration was graded (marked) to - (none). In 3 cases, tumor-infiltrating lymphocytes were remarkably increased at the delivery of a small dose of irradiation (approx. 10 Gy): 2 of them showed remarkable radiosensitivity, and the lymphocyte subpopulation of the cancer tissue was mainly composed of Leu-3a + 3b-positive and Leu-8-negative lymphocytes (helper inducer T lymphocytes or delayed-type hypersensitivity cells). On the other hand, the third one proved to be rather radioresistant, and the lymphocyte subpopulation was mainly composed of Leu-14-positive lymphocytes (B lymphocytes). These findings indicate that the analysis of the lymphocyte subpopulation infiltrating into cancer tissues at the delivery of small doses of irradiation is applicable as a predictive assay in radiotherapy of squamous cell carcinoma of the oropharynx and hypopharynx.     

Lymphocyte anergy in patients with carcinoma

All of 10 patients with colonic carcinoma and 5 with malignant melanoma of skin showed no sign of immunoreactivity against the cultured tumour cells by the lymphocyte populations residing within the tumours. More than half of these patients did show cytotoxic reactivity by their blood lymphocytes. Possible cytotoxic reactivity by the regional lymph node lymphocytes was also investigated in 57 tumour cases (44 colonic, 13 melanoma, and including 12 of the 15 examined for intrinsic lymphocyte activity). One third of the cases showed positive blood lymphocyte immunoreactivity, but in only 4 tumours (3 colonic) did the node lymphocytes show any cytotoxicity against the tumour cells. This state of anergy of intrinsic and regional lymphocytes is presumably acquired during the development of the cancer and would permit local tumour spread and metastasis to lymph nodes. Its cause has not been identified but appears to be lymphocyte inhibition rather than selective change in lymphocyte population. In particular, no special pattern can be seen in the relative proportions of T and B cells in patients'' blood, lymph node or intrinsic carcinoma lymphocytes.     

Enumeration of lymphocytes and their subpopulations identified by bacterial adherence in blood smears of patients with breast tumors

Peripheral blood lymphocyte counts were determined in 57 breast clinic patients. These patients were grouped into four clinical/pathological groups: 18 had benign breast disease, 13 had a history of breast cancer but were free of disease at the time of the study, 7 had a history of breast cancer and were free of disease at the time of the study but were on adjuvant chemotherapy, and 19 had active metastatic breast cancer. Two parameters were investigated in a double blind study: 1) the absolute lymphocyte counts and 2) the percentages of lymphocytes that bound different bacteria as markers of lymphocyte subpopulations in conventionally stained blood smears. A significant reduction in mean lymphocyte counts was demonstrated in patients with advanced disesase. A significant increase in these counts was found in patients who were free of disease following surgical treatment alone. The T₁ T₂ cells (our denomination), the subpopulation of cells responsible for specific killing in vitro and for suppression of natural cytotoxic cells, was significantly reduced in patients with advanced disease under treatment. The T/B cell ratio in all groups was the same and within normal range. These observations suggest that the observed decrease in the total lymphocytes is due not only to an overall decrease in all classes of lymphocytes but especially in those responsible for specific cell-mediated reactions. They also show that bacteria can be used as reliable reagents for the identification of lymphocyte subpopulations in blood smears.     

Possible role of prostaglandin producing monocytes in the depression of mitogenic responses of blood lymphocytes following radiation therapy

Previous studies have shown that the depression of mitogenic responses of cultured blood lymphocytes following radiation therapy can largely be explained by immunosuppressive cells. In this investigation we have shown that the addition of silica, a monocyte toxic agent, to the cultures enhance the PHA- and PPD-responses of the cells at completion of irradiation. Such an effect, however, was not noted before radiation treatment was started. Similar results were obtained by adding indomethacin, an inhibitor of prostaglandin synthesis, to the cultures. The results thus indicate that immunosuppressive monocytes which mediate their activity by prostaglandins are involved in the reductions of mitogen responses of blood lymphocytes following irradiation.     

Intrinsic radiosensitivity of normal human fibroblasts and lymphocytes after high- and low-dose-rate irradiation.

The existence of heritable radiosensitivity syndromes and clinical observations in radiotherapy patients suggests that human cellular radiosensitivity differs among individuals. We report here an in vitro study of radiosensitivity in 30 fibroblast and 29 lymphocyte cultures obtained from cancer patients and controls. In 25 cases, both fibroblasts and lymphocytes were obtained from the same donors. Fibroblasts were cultured from skin biopsy samples, and peripheral T-cell lymphocytes were cultured from blood. Clonogenic survival assays were performed by using high- and low-dose-rate irradiation; lymphocytes were in G0 phase and fibroblasts in confluent plateau phase. Various end points were calculated and compared (i.e., surviving fraction at 2 Gy, initial slope of the survival curve, and doses resulting in 10 and 1% survival, respectively). Depending on the end point, the coefficient of variation of the survival parameters ranged from 31 to 68% for lymphocytes and 21 to 41% for fibroblasts following high-dose-rate irradiation. Similar ranges were obtained after low-dose-rate irradiation. Variance analysis performed on replicate assays in cultures derived from the same patient showed that variation due to technical or sampling errors was significantly lower than variation between individuals (P = 0.00034 and 0.014 for fibroblasts and lymphocytes, respectively). No correlation was observed between the radiosensitivity of lymphocyte and fibroblast cultures derived from the same donors. We conclude that there is significant variation in normal cell radiosensitivity among individuals. On the other hand, comparisons of lymphocyte and fibroblast radiosensitivities suggest that tissue-specific characteristics, such as differentiation status, may variably modulate radiosensitivity.     

Lymphocyte counts and responses to PHA and PPD following radiation therapy for breast cancer in patients who develop recurrent disease and those who remain clinically disease-free

Peripheral blood lymphocyte counts and stimulations by PHA and PPD in vitro were examined before and up to four years after local pre- or post-operative radiation therapy of 99 patients with breast cancer. The patient material was divided into those who remained clinically disease-free during a follow up period of 4.5–7 years and those who relapsed. Radiation therapy reduced the lymphocyte counts and PPD responses to the same levels in both groups of patients; there were no essential differences in their recoveries, with the exception of a somewhat delayed recovery of the PPD-response in the patients who relapsed. PHA responses of the lymphocytes were not decreased following radiation therapy. The data indicate that these radiation induced changes of the peripheral lymphocyte population were similar both in patients who relapsed and those who remained symptom free.A group of 47 women with breast cancer that was treated by surgery only was examined similarly as a comparison. Patients from this group who developed local recurrences had higher lymphocyte counts than those who remained disease-free; patients who developed distant metastases had somewhat decreased PHA responses.     

Two-color analysis of peripheral blood lymphocytes in patients with malignant tumours after low dose half (or total) body irradiation--a pilot study

The mechanism of anti-tumour effect of low dose total body irradiation applied for non-Hodgkin's lymphoma is still unknown. Two-color analyses of peripheral blood lymphocytes from ten patients with non-Hodgkin's lymphoma or advanced cancer who received low dose total body irradiation (TBI) or half body irradiation (HBI), were performed to be a help to reveal the mechanism. The results of these analyses indicated that the proportion of helper T lymphocytes, helper-inducer T lymphocytes and cytotoxic T lymphocytes increased during TBI or HBI. On the other hand, the proportion of suppressor-inducer T lymphocytes and suppressor T lymphocytes seemed to decrease slightly. The extent of bone marrow suppression of twice-a-week TBI or HBI was clinically no problem, but in the case of thrice-a-week TBI, prolonged bone marrow suppression might occur. In the case of half body irradiated five patients who were administered with lentinan for several weeks to several months before TBI or HBI, the pre-HBI value of the proportion of cytotoxic T lymphocyte fraction was found to be high, and the increment of the proportion of helper T lymphocyte was larger than that of non-lentinan treated cases.     

Application of the immunohistochemical method as predictive assay in radiotherapy of squamous cell carcinoma

We analysed various subsets of lymphocytes infiltrated into cancer tissues from 15 patients (10 cases of oropharyngeal cancer and 5 of hypopharyngeal cancer) at pretreatment and at about 10 Gy and 30 Gy of irradiation by the method of biotin-avidin-horseradish peroxidase using mouse monoclonal antibodies. In 3 cases, tumor infiltrating lymphocytes were remarkably increased at the delivery of small dose of irradiation: Two of them showed remarkable radiosensitivity, and the lymphocyte subpopulation of the cancer tissue was mainly composed of Leu3a + 3b positive, Leu-8 negative and Leu-HLA-DR positive lymphocytes (activated helper inducer T lymphocytes). On the other hand, the third one proved to be rather radioresistant and the lymphocyte subpopulation was mainly composed of Leu-14 positive lymphocytes (B lymphocytes). These findings indicate that the analysis of the lymphocyte subpopulation infiltrating into cancer tissues at the delivery of small dose of irradiation is applicable as a predictive assay in radiotherapy of squamous cell carcinoma of oropharynx and hypopharynx.     

A radiation-induced gene expression signature as a tool to predict acute radiotherapy-induced adverse side effects

The majority of patients tolerate radiotherapy well, but some of them suffer from severe side effects. To find genes possibly predictive for radiosensitivity, mRNA profiles were generated before and 6 h after in vitro irradiation with 5 Gy. We analyzed lymphocytes from four head and neck and eight breast cancer patients with strong acute radiation toxicity and from 12 matching normal reacting patients in a blind study. Expression was also measured in lymphocyte subpopulations and Epstein–Barr transformed lymphocytes. Radiation response in whole lymphocyte populations was most similar to that of B cells. In peripheral blood lymphocytes of all patients; 153 genes were identified which were statistically significantly altered by a fold change of more than 50% by irradiation. The signatures of radio-responsive genes differed tremendously between primary and transformed cells. Pathway analysis revealed genes involved in p53 signalling, cell cycle control and apoptosis in response to radiation in primary lymphocytes. In these cells, a set of 67 radiation-induced genes was identified capable of differentiating between severe radiosensitive and normal reacting patients. More than one third of such classifying genes belong to the group of apoptosis or cell cycle regulating genes. The classifying potential of the expression signature has now to be validated in further patient cohorts.     

Effect of radiation therapy and in vitro x-ray exposure on lymphocyte subpopulations and their functions

Radiation treatment of breast cancer patients (45.0 Gy) profoundly affected the peripheral blood lymphocytes. The number of these cells was markedly reduced with non-T-cells being more extensively depleted than T-cells immediately after radiation. The long-lasting lymphopenia, on the other hand, was mainly due to reduced number of T-cells. Antigen and mitogen stimulability, MLC reactivity, pokeweed (PWM)-induced immunoglobulin (Ig) production in vitro, and different cytotoxic functions decreased. Depletion of lymphocytes largely restored the radiation-depressed lymphocyte reactivity. The effects of in vitro exposure of blood lymphocytes to x-rays were similar to those seen after radiotherapy. Non-T-cells and T-cells with Fc-receptors for IgG were relatively radiosensitive. This latter observation agreed well with demonstrated increase of PWM-induced Ig synthesis after in vitro exposure to x-rays. T-suppressor cells defined by monoclonal antibodies were, however, radioresistant. The cytotoxic functions were reduced. No correlations were found between the pretreatment immunological status or the extent of radiation-induced immunological suppression, respectively, and prognosis.     

Depressed in vitro peripheral blood lymphocyte response to mitogens in cancer patients: the role of suppressor cells.

The reactivity of peripheral blood lymphocytes from patients with advanced malignancy was assessed by mitogen-induced stimulation of protein synthesis as measured by 3H-leucine incorporation. It was confirmed that the lymphocyte response of patients was depressed. Furthermore, the lymphocytes of 15 out of 27 cancer patients, selected because of their low responses, inhibited the reactivity of normal lymphocytes in co-cultures. The lymphocytes from one patient with Hodgkin's disease were also inhibitory. In contrast, lymphocytes from healthy subjects, patients with chronic lymphocytic leukaemia, lymphosarcoma or multiple myeloma caused no suppression. Experiments with purified cell populations from patients with carcinoma indicated that purified T cells responded to mitogens while unseparated lymphocytes failed to respond and that the inhibitory activity was due to adherent cells, presumably monocytes. There was no evidence for B-cell-mediated suppression. However, in two cases inhibition was caused by isolated T cells of the patients and not by adherent cells. These experiments suggested that one mechanism for the depression of cell-mediated immunity seen in patients with advanced cancer may be the nonspecific suppresssion of certain T-cell functions by circulating monocytes.