ChemoImmunoModulation: immune regulation by the antineoplastic chemotherapeutic agents.

Since 1948, when Farber et al. introduced aminopterin, the first chemotherapeutic agent, more than 100 such agents have come into use. Initially, antitumor chemotherapies were thought to produce only antiproliferative or cytotoxic effects on dividing tumor cells as it was often associated with the damage to healthy tissues and the development of resistant tumor clones. However, that view has been changing as a consequence of recent demonstrations that several antineoplastic drugs, even at low doses, have antiangiogenic and sometimes immunomodulating effects. In addition, new studies indicate that lowering the dose of conventional cytotoxic agents and combining chemotherapy with other modalities may not only decrease the toxicity of conventional chemotherapy, but also up-regulate the efficacy of different anticancer therapies. Giving chemotherapy in this manner has several potential advantages, including impediment of the onset of mutation-dependent mechanisms of acquired drug resistance and increase in the efficacy and durability of combinatorial therapeutic modalities. Certain "immunogenic" forms of cancer chemotherapy may cause indirect activation of immune cells due to the accessibility of tumor antigens and certain "danger" signals. Furthermore, new findings indicate that several chemotherapeutic agents can directly activate immune cells when used in ultra low noncytotoxic concentrations, the new phenomenon that was termed chemoimmunomodulation. The goal of this review is to analyze the immune modulating properties of antineoplastic chemotherapeutic agents and present new evidence of the immunostimulating potentials of several agents used in low and ultra low nontoxic doses. Therapeutic potentials of combined chemo-immunotherapeutic regimens have been extensively reviewed in a variety of recent publications and will not be discussed.

Comparative analysis of CD1a, S-100, CD83, and CD11c human dendritic cells in normal, premalignant, and malignant tissues.

A number of antibodies that recognize human dendritic cells (DC) have been identified. The main aim of this study was to compare and contrast different antigen retrieval techniques using both enzymatic and non-enzymatic treatments in order to determine the expression and distribution of several DC markers on formalin-fixed, paraffin-embedded tissues. Normal human lung, oral epithelial hyperplasia lesions, oral squamous cell carcinoma, and prostate adenocarcinoma tissues were evaluated using a panel of DC specific antibodies. The results of immunohistochemical staining for CD83, CD1a, CD11c, and S-100 DC markers were compared following the different antigen retrieval approaches. The overall best results for the analysis of tumor-associated DC were obtained with the enzymatic methods. Protease XXIV digestion was determined to be essential for detection of S-100 and CD11c positive DC, whereas trypsin and pepsin were required for the recognition of CD1a and CD83 expressing tumor-associated DC. These results could be easily adapted for routine practice and should be useful for characterization of the DC system in cancer patients for both diagnostic and prognostic purposes. In addition, standardized procedures for evaluating different subpopulations of tumor-associated DC should bring new insights in understanding of DC-tumor cell interaction.

Mechanisms of dendritic cell-induced T cell proliferation in the primary MLR assay.

DC are unique antigen presenting cells, and their ability to induce proliferation of T cells in a mixed leukocyte reaction (MLR) assay is commonly used for the evaluation of their function. To determine the mechanisms involved in DC-induced T cell activation in a primary MLR assay, a variety of different agents were examined in this study that interfere with DNA synthesis, membrane organization, protein synthesis, and maturation induced by bacterial products. While only live DC were able to induce T cell proliferation in the MLR assay, irradiation of DC did not influence their stimulatory capacity. Fixation of DC membrane with paraformaldehyde resulted in a loss of DC capacity to induce T cell proliferation demonstrating that physical organization of the plasma membranes plays an important role in the induction of T cell activation. In addition, the pretreatment of DC with cycloheximide revealed that protein synthesis was not critical for the ability of DC to activate T cells. Finally, Staphylococcus aureus-mediated activation of DC significantly increased T cell proliferation and this effect was not dependent on IL-12 production of DC since DC generated from IL-12 knockout mice were not different from wild type DC. In summary, these data suggest that DC membrane structures are responsible for the antigen presentation and co-stimulation and play a key role in T cell recognition and activation by DC.

Identification of delta- and mu-type opioid receptors on human and murine dendritic cells.

The purpose of this study was to evaluate mu- and delta-opioid receptors (OR) on human and murine dendritic cells (DC). Expression of mu- and delta-OR mRNA on DC was demonstrated by RT-PCR. The immunocytochemical and Western blot analyses revealed the expression of OR protein in DC. Radioreceptor assay demonstrated the specific saturated temperature-dependent binding of [3H]-labeled opioid ligand on DC and B(max)=2.8+/-0.3 fmol/10(6) cells and K(D)=4.8+/-1.0 nM were calculated by a Scatchard analysis. Finally, OR ligands DADLE and DAGO dose-dependently modulated the capacity of DC to induce T cell proliferation in an MLR assay. Importantly, expression of functional OR on DC was significantly increased upon TNF-alpha-induced DC maturation. Thus, these data suggest a new mechanism of opioid-dependent neuroendocrine immunomodulation.

Tumor necrosis factor-alpha-promoted expression of Bcl-2 and inhibition of mitochondrial cytochrome c release mediate resistance of mature dendritic cells to melanoma-induced apoptosis.

Melanoma escapes host defenses through a variety of means, including the elimination of immune effector cells within the tumor microenvironment. We have reported recently that murine and human tumors including melanoma induce premature apoptosis of dendritic cells both in vitro and in vivo. In this study, we have demonstrated that overexpression of the Bcl-2 protein family member Bcl-xL rescued murine dendritic cells (DCs) from melanoma-induced death in vitro. Another successful protection approach was tumor necrosis factor (TNF)-alpha-promoted sustained expression of the antiapoptotic protein Bcl-2 within dendritic cells. This effect of TNF-alpha was mediated by inhibition of mitochondrial cytochrome c release. Thus, both Bcl-xL and Bcl-2 enhance survival of dendritic cells within the tumor microenvironment. In addition, mature DCs were more resistant to melanoma-induced apoptosis than immature dendritic cells. This finding suggests a stage-dependent sensitivity of DCs to tumor-induced cell death. We conclude that: (a) mature DCs might be more suitable for the use of cancer vaccination; and (b) Bcl-2 protein family members such as Bcl-xL and Bcl-2 rescue DCs from tumor-induced premature apoptosis.

Neuroblastoma-derived gangliosides inhibit dendritic cell generation and function.

Neuroblastoma (NB), a tumor of the sympathetic nervous system, is the most common extracranial solid tumor in children. NB-derived gangliosides inhibit the functional activity of T and natural killer cells, contribute to tumor-induced bone marrow suppression, and cause multiple alterations of hematopoiesis, resulting in pancytopenia. However, the role of gangliosides in the regulation of dendritic cell (DC) generation (dendropoiesis) has not been studied. Using murine and human NB cell lines, we demonstrated that coincubation of murine bone marrow progenitors or human CD34+ progenitor cells with NB cells resulted in a significant inhibition of dendropoiesis in vitro up to 90%. The number of DCs was assessed by FACScan determination of CD83+ or CD11c+ cells coexpressing MHC class II and CD86 molecules. In addition, inhibition of antigen-presenting properties of DCs cultured in the presence of NB cells was observed in allogeneic mixed leukocyte reaction (33,508 +/- 1,613 cpm for control DCs versus 17,428 +/- 152 cpm for NB-treated DCs; P < 0.05). Treatment of NB cells with 10 microM DL-threo-1-phenyl-2-decanolylamine-3-morpholino-1-propanol HCl, an inhibitor of glucosylceramide synthase, markedly abrogated ganglioside synthesis and was accompanied by blockade of NB ability to inhibit dendropoiesis. Furthermore, purified gangliosides added to DC cultures significantly inhibited DC generation. The percentage of CD83+ cells decreased from 51.8 +/- 6.1% in the control group to 12.9 +/- 2.7% in cultures treated with GD2 (P < 0.05). Thus, our results demonstrate that NB-derived gangliosides inhibit the generation of functionally active DCs and may play a role in tumor-induced immunosuppression and subsequent tumor escape from immune recognition and elimination.

Human small cell lung carcinoma and carcinoid tumor regulate dendritic cell maturation and function.

The induction of apoptosis in dendritic cells (DC) is a key mechanism by which tumors escape immune recognition and elimination. In fact, a number of studies have showed the correlation between the number of DC within the tumor and the clinical prognosis, suggesting that increased infiltration of tumor tissue by DC was associated with better patient survival and low incidence of metastatic disease. We compared the number of DC and their distribution pattern in human small-cell lung carcinoma and bronchial carcinoid tumor (CT) tissues. Immunohistochemical analysis revealed the presence of cells expressing DC markers CD1a and CD83 in small-cell lung carcinoma tissues and the complete absence of these cells in CT samples. Next, we examined whether human lung tumor cells produce soluble factors that inhibit differentiation of hematopoietic precursors into mature DC. The addition of small-cell lung carcinoma-conditioned medium to CD34+ precursor cell cultures significantly inhibited colony-forming units of DC formation when compared with nontreated control DC cultures. Furthermore, DC generation and differentiation was completely abrogated in CD34+ cell cultures treated with CT-conditioned medium, suggesting that CT-derived factors blocked CD34+ cell differentiation into DC or induced their apoptosis. Finally, flow cytometry analysis of cultured DC confirmed these results. Thus, analysis of our data suggests that human lung tumors produce factors that inhibit DC generation or maturation and may also induce apoptotic death of DC precursors in vitro.

Human prostate cancer regulates generation and maturation of monocyte-derived dendritic cells.

BACKGROUND: The progression of prostate cancer is accompanied by a marked suppression of the immune system, including the apoptotic death of dendritic cells (DC) responsible for the induction of antitumor immunity. In this study, we evaluated whether prostate cancer might inhibit DC generation and maturation in vitro. METHODS: DC were generated from peripheral blood monocytes in the presence of the human prostate cell line LNCaP or nonmalignant cells, and characterized by light microscopy, FACScan analysis, and ability to stimulate T-cell proliferation. RESULTS: Prostate cancer significantly inhibited the conversion of monocytes into DC, which was assessed by the expression of DC markers CD1a and CD83. These cells were weak stimulators of T-cell proliferation, suggesting that DC generated in the prostate cancer microenvironment are functionally inhibited. CONCLUSIONS: Prostate cancer not only kills mature DC, but also inhibits their generation and maturation, resulting in decreased production of antigen-presenting cells and inhibition of their functional activity.

TNF-alpha protects dendritic cells from prostate cancer-induced apoptosis.

We have recently shown that human prostate cancer (PCa) cells induced apoptotic death of the most potent antigen-presenting cells, dendritic cells (DC), which are responsible for the induction of specific antitumor immune responses. Here we have evaluated the effect of murine PCa cells RM-1 on the survival of immature and tumor necrosis factor-alpha (TNF-alpha)-stimulated mature DC. PCa cells and DC were co-incubated for 24-48 h and DC apoptosis was assessed by morphologic criteria, Annexin V assay, and TUNEL staining. We have shown that co-incubation of RM-1 cells with DC is accompanied by an increased level of DC apoptosis, which was mediated by decreased expression of anti-apoptotic protein Bcl-2. Stimulation of DC maturation by TNF-alpha resulted in increased resistance of DC to PCa-induced apoptosis. In TNF-alpha treated mature DC, but not in immature DC, the expression of Bcl-2 was not blocked after exposure to RM-1-derived factors. Thus, these data suggest that TNF-alpha-induced maturation of DC increases their resistance to PCa induced apoptosis. This is likely to be due to the stabilizing of the expression of anti-apoptotic protein Bcl-2. The difference in the sensitivity of mature and immature DC to PCa-induced cell death should be considered during the design of DC-based clinical trials for PCa patients.Prostate Cancer and Prostatic Diseases (2001) 4, 221-227.

Transduction of dendritic cells with Bcl-xL increases their resistance to prostate cancer-induced apoptosis and antitumor effect in mice.

We have shown that prostate cancer (PCa) causes apoptosis of dendritic cells (DC), which might block the development of specific antitumor immune responses. Analysis of murine prostatic carcinoma tissues revealed the significant decrease in intratumoral DC number during tumor progression. We demonstrated that the cytokine-mediated increase in DC survival was accompanied by an elevated expression of the anti-apoptotic protein Bcl-xL. Next, we evaluated the resistance to tumor-induced apoptosis and the antitumor efficiency of genetically engineered DC overexpressing Bcl-xL. DC were transduced with an adenoviral vector encoding the murine Bcl-xL gene and injected intratumorally. Data analysis revealed that treatment of PCa-bearing mice with Bcl-xL-transduced DC resulted in significant inhibition of tumor growth compared with the administration of nontransduced DC. Thus, our data suggest that the protection of DC from PCa-induced apoptosis might significantly increase the efficacy of DC-based therapies in cancer even in the absence of available tumor-specific Ags.

Cytokine-mediated protection of human dendritic cells from prostate cancer-induced apoptosis is regulated by the Bcl-2 family of proteins.

Prostate cancer is the most common cancer in men in the United States, and second in cancer-induced mortality. It is likely that tumour-induced immunosuppression is one of the reasons for low treatment efficacy in patients with advanced prostate cancer. It has been recently demonstrated that prostate cancer tissue is almost devoid of dendritic cells (DC), the major antigen-presenting cells responsible for the induction of specific antitumour immune responses. In this study, we have tested the hypothesis that prostate cancer induces progressive suppression of the DC system. We found that co-incubation of human DC with three prostate cancer cell lines led to the high levels of premature apoptosis of DC, which were significantly higher than in DC cultures co-incubated with normal prostate cells or blood leucocytes. Stimulation of DC for 24 hours with CD40 ligand (CD154), IL-12 or IL-15 prior to their co-incubation with prostate cancer cells resulted in a significant increase in DC survival in the tumour microenvironment. Furthermore, activation of DC with these cytokines was also accompanied by increased expression of the anti-apoptotic protein Bcl-x(L) in DC, suggesting a possible mechanism involved in DC protection from apoptotic death. In summary, our data demonstrate that prostate cancer induces active elimination of DC in the tumour microenvironment. Stimulation of DC by CD154, IL-12 or IL-15 leads to an increased expression of the anti-apoptotic protein Bcl-x(L) and increased resistance of DC to prostate cancer-induced apoptosis. These results suggest a new mechanism of tumour escape from immune recognition and demonstrate the cytokine-based approaches which might significantly increase the efficacy of DC-based therapies for cancer.

Tumor's other immune targets: dendritic cells.

The induction of apoptosis in T cells is one of several mechanisms by which tumors escape immune recognition. We have investigated whether tumors induce apoptosis in dendritic cells (DC) by co-culture of murine or human DC with different tumor cell lines for 4-48 h. Analysis of DC morphological features, JAM assay, TUNEL, caspase-3-like and transglutaminase activity, Annexin V binding, and DNA fragmentation assays revealed a time- and dose-dependent induction of apoptosis in DC by tumor-derived factors. This finding is both effector and target specific. The mechanism of tumor-induced DC apoptosis involved regulation of Bcl-2 and Bax expression. Double staining of both murine and human tumor tissues confirmed that tumor-associated DC undergo apoptotic death in vivo. DC isolated from tumor tissue showed significantly higher levels of apoptosis as determined by TUNEL assay when compared with DC isolated from spleen. These findings demonstrate that tumors induce apoptosis in DC and suggest a new mechanism of tumor escape from immune recognition. DC protection from apoptosis will lead to improvement of DC-based immunotherapies for cancer and other immune diseases.

T-lymphocyte activation increases hypothalamic and amygdaloid expression of CRH mRNA and emotional reactivity to novelty.

Stimulation of T-cells with staphylococcal enterotoxin B (SEB) significantly elevates interleukin-2 (IL-2) and contemporaneous activation of the hypothalamic-pituitary-adrenal (HPA) axis and c-fos in the paraventricular nucleus (PVN) of BALB/cByJ mice. Such neural signaling may promote cognitive and emotional adaptation before or during infectious illness. Because corticotropin-releasing hormone (CRH) is an anxiogenic neuropeptide that may mediate the stressor-like effects of immunological stimuli, we measured neuronal CRH mRNA alterations in mice challenged with SEB. Increased CRH mRNA levels were observed in the PVN and central nucleus of the amygdala (ceA) 4-6 hr after SEB administration. This was associated with plasma ACTH increases, which could be abrogated by the systemic administration of anti-CRH antiserum. Additional experiments did not support a role for IL-2 or prostaglandin synthesis in activating the HPA axis. Behavioral experiments testing for conditioned taste aversion did not confirm that SEB challenge promotes malaise. However, consistent with the notion that central CRH alterations induced by SEB may affect emotionality (e.g., fear), SEB challenge augmented appetitive neophobia in a context-dependent manner, being marked in a novel and stressful environment. It is hypothesized that immunological stimuli generate a cascade of events that solicit integrative neural processes involved in emotional behavior. As such, these data support the contention that affective illness may be influenced by immunological processes and the production of cytokines and are consistent with other evidence demonstrating that autoimmune reactivity is associated with enhanced emotionality.

Apoptosis induced in T cells by human neuroblastoma cells: role of Fas ligand.

The CD95/CD95L (Fas/Fas ligand) receptor/ligand system plays an important role in regulation of cell survival and induction of a programmed cell death. It is also involved in regulation of effector phase of T and NK cell cytotoxicity, establishment of immune privilege sites, and tumor escape from immune recognition. In this study, we assessed expression of CD95L in tumors obtained from patients with neuroblastoma (NB) and in established NB cell lines. We measured the presence of intratumoral T cell infiltrates and T cell survival in tumor tissue samples. High levels of apoptosis were observed in tumor-associated lymphocytes as well as in Jurkat T cells cocultured with NB cells in vitro. T cell death was reduced after treatment of NB cells (in vitro) with antibody to FAS ligand (FasL). Overall, our data suggest that NB-induced apoptosis of Fas-sensitive Jurkat T cells is mediated by functional FasL expressed on NB and Fas/FasL interaction may be responsible for the elimination of T cells in the NB microenvironment.

Hypothalamic-pituitary-adrenal activation by the bacterial superantigen staphylococcal enterotoxin B: role of macrophages and T cells.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen which stimulates T cells bearing the V beta 8 motif on the T-cell receptor. This stimulation is MHC class II dependent, and in vivo results in a rapid and pronounced T-cell cytokine response. Based on previous evidence that SEB stimulates corticosterone production in BALB/c mice, which possess a high percentage of V beta 8+ T cells, we explored the effects of SEB on the hypothalamic-pituitary-adrenal (HPA) axis and identified the peripheral immunologic cellular requirements for these effects. Administration of SEB stimulates corticosterone in a dose-dependent manner, with peak production of corticosterone occurring by 2 h after intraperitoneal challenge with 50 micrograms SEB. Challenge with staphylococcal enterotoxin A, which activates V beta 3+ and V beta 11+ T cells (deleted during ontogenesis in BALB/c mice), did not increase ACTH or corticosterone production. Furthermore, SEB challenge increased plasma ACTH, which accounted for the increased plasma corticosterone, and increased the expression of c-fos in the PVN region of the hypothalamus. In vivo elimination of macrophages did not prevent the corticosterone response to SEB, suggesting that pituitary-adrenal activation does not require macrophages. However, when mice were pretreated with the T-cell immunosuppressant cyclosporin A, the significantly increased ACTH and corticosterone production in response to SEB was dramatically attenuated. These results demonstrate that bacterial superantigens can stimulate the HPA axis, and that functional T cells may play an obligatory role in this effect.

Mitogen-stimulated interleukin-2 production in never-medicated, first-episode schizophrenic patients. The influence of age at onset and negative symptoms.

BACKGROUND: Decreased interleukin-2 (IL-2) production is characteristic of active autoimmune diseases and has previously been reported in patients with schizophrenia. We attempted to replicate this finding in never-medicated schizophrenic patients and examine the possible correlation between IL-2 production and clinical variables. METHODS: The production of IL-2 was measured in equal numbers (N = 33) of DSM-III-R-diagnosed schizophrenic patients and controls who were matched for age, race, and gender. Patients were also assessed for positive, negative, and depressive symptoms. RESULTS: The production of IL-2 was significantly lower in patients than in controls. There was a significant positive correlation between IL-2 production and age at onset, and significant negative correlation between IL-2 production and negative symptom scores. In multivariate analyses, the predictive power was stronger for age at onset than for negative symptoms. Positive or depressive symptoms were unrelated to IL-2 production. CONCLUSIONS: Our finding of low IL-2 production in neuroleptic-native schizophrenic patients confirms that this finding is not confounded by medications. The correlation of low IL-2 production with younger age at onset suggests that this may be a marker for a subtype of the illness or for severity.

An association between anti-hippocampal antibody concentration and lymphocyte production of IL-2 in patients with schizophrenia.

Serum concentrations of anti-hippocampal antibodies and in vitro production of the lymphokine interleukin-2 (IL-2) in response to phytohaemagglutinin (PHA) stimulation were determined using an enzyme immunoassay in 49 schizophrenic patients and 41 healthy controls. Decrease in IL-2 production, a finding frequently associated with many autoimmune diseases, was associated with an elevation in anti-hippocampal antibody optical density (AHA-OD) in schizophrenic patients. Although some control subjects had elevated antibody levels, this elevation was not associated with decreased IL-2 production. Low IL-2 production is well known to be a state marker associated with active autoimmune disease. We suggest that production of hippocampal antibody is a trait marker of vulnerability to autoimmune diseases. Thus, our finding of low IL-2 production in patients with high concentrations of hippocampal antibody is compatible with the possibility that such patients have an ongoing autoimmune process.