Galina V. Shurin

Serum interleukin-6 concentration in schizophrenia: Elevation associated with duration of illness

Using an enzyme immunoassay (ELISA), we measured serum interleukin-6 (IL-6) concentration in 128 schizophrenic patients (24 of whom were never medicated) and in 110 normal control subjects. Mean serum IL-6 concentration was significantly higher in the schizophrenic patients as compared with the control subjects (p = 0.009). Comparisons within the patient group revealed that serum IL-6 was significantly correlated with duration of illness (r = 0.32, p = 0.0004). After covariation for duration of illness, there was no relationship between IL-6 levels and the production of autoantibodies, clinical state, or medication status. Thus, elevated serum IL-6 levels in schizophrenia develop during the course of illness and may be related to treatment or to disease progression.

Loss of New Chemokine CXCL14 in Tumor Tissue Is Associated with Low Infiltration by Dendritic Cells (DC), while Restoration of Human CXCL14 Expression in Tumor Cells Causes Attraction of DC Both In Vitro and In Vivo

Breast and kidney-expressed chemokine (BRAK) CXCL14 is a new CXC chemokine with unknown function and receptor selectivity. The majority of head and neck squamous cell carcinoma (HNSCC) and some cervical squamous cell carcinoma do not express CXCL14 mRNA, as opposed to constitutive expression by normal oral squamous epithelium. In this study, we demonstrate that the loss of CXCL14 in HNSCC cells and at HNSCC primary tumor sites was correlated with low or no attraction of dendritic cell (DC) in vitro, and decreased infiltration of HNSCC mass by DC at the tumor site in vivo. Next, we found that recombinant human CXCL14 and CXCL14-positive HNSCC cell lines induced DC attraction in vitro, whereas CXCL14-negative HNSCC cells did not chemoattract DC. Transduction of CXCL14-negative HNSCC cell lines with the human CXCL14 gene resulted in stimulation of DC attraction in vitro and increased tumor infiltration by DC in vivo in chimeric animal models. Furthermore, evaluating the biologic effect of CXCL14 on DC, we demonstrated that the addition of recombinant human CXCL14 to DC cultures resulted in up-regulation of the expression of DC maturation markers, as well as enhanced proliferation of allogeneic T cells in MLR. Activation of DC with recombinant human CXCL14 was accompanied by up-regulation of NF-κB activity. These data suggest that CXCL14 is a potent chemoattractant and activator of DC and might be involved in DC homing in vivo.

Chemotherapeutic Agents in Noncytotoxic Concentrations Increase Antigen Presentation by Dendritic Cells via an IL-12-Dependent Mechanism

Antineoplastic chemotherapeutic agents may indirectly activate dendritic cells (DCs) by inducing the release of “danger” signals from dying tumor cells. Whereas the direct cytotoxic or inhibitory effect of conventional chemotherapy on DCs has been reported, modulation of DC function by chemotherapeutic agents in low noncytotoxic concentrations has not yet been investigated. We have tested the effects of different classes of antineoplastic chemotherapeutic agents used in low noncytotoxic concentrations on the Ag-presenting function of DCs. We revealed that paclitaxel, doxorubicin, mitomycin C, and methotrexate up-regulated the ability of DCs to present Ags to Ag-specific T cells. Stimulation of DC function was associated with the up-regulation of expression of Ag-processing machinery components and costimulatory molecules on DCs, as well as increased IL-12p70 expression. However, the ability of DCs treated with paclitaxel, methotrexate, doxorubicin, and vinblastine to increase Ag presentation to Ag-specific T cells was abolished in DCs generated from IL-12 knockout mice, indicating that up-regulation of Ag presentation by DCs is IL-12-dependent and mediated by the autocrine or paracrine mechanisms. At the same time, IL-12 knockout and wild-type DCs demonstrated similar capacity to up-regulate OVA presentation after their pretreatment with low concentrations of mitomycin C and vincristine, suggesting that these agents do not utilize IL-12-mediated pathways in DCs for stimulating Ag presentation. These findings reveal a new mechanism of immunopotentiating activity of chemotherapeutic agents—a direct immunostimulatory effect on DCs (chemomodulation)—and thus provide a strong rationale for further assessment of low-dose chemotherapy given with DC vaccines for cancer treatment.

Intratumoral cytokines/chemokines/growth factors and tumor infiltrating dendritic cells: friends or enemies?

The tumor microenvironment consists of a variable combination of tumor cells, stromal fibroblasts, endothelial cells and infiltrating leukocytes, such as macrophages, T lymphocytes, and dendritic cells. A variety of cytokines, chemokines and growth factors are produced in the local tumor environment by different cells accounting for a complex cell interaction and regulation of differentiation, activation, function and survival of multiple cell types. The interaction between cytokines, chemokines, growth factors and their receptors forms a comprehensive network at the tumor site, which is primary responsible for overall tumor progression and spreading or induction of antitumor immune responses and tumor rejection. Although the general thought is that dendritic cells are among the first cells migrating to the tumor site and recognizing tumor cells for the induction of specific antitumor immunity, the clinical relevance of dendritic cells at the site of the tumor remains a matter of debate regarding their role in the generation of successful antitumor immune responses in human cancers. While several lines of evidence suggest that intratumoral dendritic cells play an important role in antitumor immune responses, understanding the mechanisms of dendritic cell/tumor cell interaction and modulation of activity and function of different dendritic cell subtypes at the tumor site is incomplete. This review is limited to discussing the role of intratumoral cytokine network in the understanding immunobiology of tumor-associated dendritic cells, which seems to possess different regulatory functions at the tumor site.

Dendritic Cells in the Cancer Microenvironment

The complexity of the tumor immunoenvironment is underscored by the emergence and discovery of different subsets of immune effectors and regulatory cells. Tumor-induced polarization of immune cell differentiation and function makes this unique environment even more intricate and variable. Dendritic cells (DCs) represent a special group of cells that display different phenotype and activity at the tumor site and exhibit differential pro-tumorigenic and anti-tumorigenic functions. DCs play a key role in inducing and maintaining the antitumor immunity, but in the tumor environment their antigen-presenting function may be lost or inefficient. DCs might be also polarized into immunosuppressive/tolerogenic regulatory DCs, which limit activity of effector T cells and support tumor growth and progression. Although various factors and signaling pathways have been described to be responsible for abnormal functioning of DCs in cancer, there are still no feasible therapeutic modalities available for preventing or reversing DC malfunction in tumor-bearing hosts. Thus, better understanding of DC immunobiology in cancer is pivotal for designing novel or improved therapeutic approaches that will allow proper functioning of DCs in patients with 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. J. Leukoc. Biol. 66: 336–344; 1999.

Antitumor Effect of Paclitaxel Is Mediated by Inhibition of Myeloid-Derived Suppressor Cells and Chronic Inflammation in the Spontaneous Melanoma Model

The antitumor effects of paclitaxel are generally attributed to the suppression of microtubule dynamics resulting in defects in cell division. New data demonstrated that in ultralow noncytotoxic concentrations, paclitaxel modulated in immune cells in vitro the activity of small Rho GTPases, the key regulators of intracellular actin dynamics. However, the immunomodulatory properties of paclitaxel in vivo have not been evaluated. In this study, using the ret transgenic murine melanoma model, which mimics human cutaneous melanoma, we tested effects of ultralow noncytotoxic dose paclitaxel on functions of myeloid-derived suppressor cells (MDSCs), chronic inflammatory mediators, and T cell activities in the tumor microenvironment in vivo. Administration of paclitaxel significantly decreased accumulation and immunosuppressive activities of tumor-infiltrating MDSCs without alterations of the bone marrow hematopoiesis. This was associated with the inhibition of p38 MAPK activity, TNF-α and production, and S100A9 expression in MDSCs. The production of mediators of chronic inflammation in the tumor milieu also was diminished. Importantly, reduced tumor burden and increased animal survival upon paclitaxel application was mediated by the restoration of CD8 T cell effector functions. We suggest that the ability of paclitaxel in a noncytotoxic dose to block the immunosuppressive potential of MDSCs in vivo represents a new therapeutic strategy to downregulate immunosuppression and chronic inflammation in the tumor microenvironment for enhancing the efficacy of concomitant anticancer therapies.

Low-Dose Paclitaxel Prior to Intratumoral Dendritic Cell Vaccine Modulates Intratumoral Cytokine Network and Lung Cancer Growth

Purpose: The main goal of this study was to provide the “proof-of-principle” that low-dose paclitaxel is able to change the tumor microenvironment and improve the outcome of intratumoral dendritic cell vaccine in a murine lung cancer model. Experimental Design: We evaluated the antitumor potential and changes in the intratumoral milieu of a combination of low-dose chemotherapy and dendritic cell vaccine in the Lewis lung carcinoma model in vivo. Results: The low-dose paclitaxel, which induced apoptosis in ∼10% of tumor cells, was not toxic to bone marrow cells and dendritic cells and stimulated dendritic cell maturation and function in vitro. Although tumor cells inhibited dendritic cell differentiation in vitro, this immunosuppressive effect was abrogated by the pretreatment of tumor cells with low-dose paclitaxel. Based on these data, we next tested whether pretreatment of tumor-bearing mice with low-dose paclitaxel in vivo would improve the antitumor potential of dendritic cell vaccine administered intratumorally. Significant inhibition of tumor growth in mice treated with low-dose paclitaxel plus intratumoral dendritic cell vaccine, associated with increased tumor infiltration by CD4+ and CD8+ T cells and elevated tumor-specific IFN-γ production by draining lymph node cells, was revealed. Using a novel intratumoral microdialysis technique and Luminex technology for collecting and characterizing soluble factors released within the tumor bed for several days in live freely moving animals, we showed that low-dose paclitaxel altered the cytokine network at the tumor site. Conclusions: Our data indicate that low-dose chemotherapy before intratumoral delivery of dendritic cells might be associated with beneficial alterations of the intratumoral microenvironment and thus support antitumor immunity.

Chemomodulation of human dendritic cell function by antineoplastic agents in low noncytotoxic concentrations

The dose-delivery schedule of conventional chemotherapy, which determines its efficacy and toxicity, is based on the maximum tolerated dose. This strategy has lead to cure and disease control in a significant number of patients but is associated with significant short-term and long-term toxicity. Recent data demonstrate that moderately low-dose chemotherapy may be efficiently combined with immunotherapy, particularly with dendritic cell (DC) vaccines, to improve the overall therapeutic efficacy. However, the direct effects of low and ultra-low concentrations on DCs are still unknown. Here we characterized the effects of low noncytotoxic concentrations of different classes of chemotherapeutic agents on human DCs in vitro. DCs treated with antimicrotubule agents vincristine, vinblastine, and paclitaxel or with antimetabolites 5-aza-2-deoxycytidine and methotrexate, showed increased expression of CD83 and CD40 molecules. Expression of CD80 on DCs was also stimulated by vinblastine, paclitaxel, azacytidine, methotrexate, and mitomycin C used in low nontoxic concentrations. Furthermore, 5-aza-2-deoxycytidine, methotrexate, and mitomycin C increased the ability of human DCs to stimulate proliferation of allogeneic T lymphocytes. Thus, our data demonstrate for the first time that in low noncytotoxic concentrations chemotherapeutic agents do not induce apoptosis of DCs, but directly enhance DC maturation and function. This suggests that modulation of human DCs by noncytotoxic concentrations of antineoplastic drugs, i.e. chemomodulation, might represent a novel approach for up-regulation of functional activity of resident DCs in the tumor microenvironment or improving the efficacy of DCs prepared ex vivo for subsequent vaccinations.

Inhibition of CD40 expression and CD40-mediated dendritic cell function by tumor-derived IL-10

As CD40 plays a key role in both antitumor immunity and DC maturation, we have studied the regulation of its expression during DC hematopoiesis (dendropoiesis) in vitro and in vivo in the tumor microenvironment. Using MC38 colon adenocarcinoma tumor models, we have demonstrated that DCs generated in vitro from bone marrow precursors obtained from tumor‐bearers have significantly lower expression of CD40 molecules compared to DCs generated from tumor‐free mice. Furthermore, CD40 expression on DCs isolated from the spleens of tumor‐bearing mice was also significantly reduced, suggesting that tumor‐derived factors inhibit CD40 expression on DCs during dendropoiesis both in vitro and in vivo. Interestingly, CD40 ligation on DCs generated from tumor‐bearers did not result in inducible expression of IL‐12 protein or IL‐12 p40 mRNA. However, Staphylococcus aureus–induced IL‐12 production by DCs was not altered in tumor‐bearers, confirming that inhibition of IL‐12 production by DCs generated in vitro from tumor‐bearing mice was due to reduced expression of CD40 on DCs. We have also shown that MC38 tumor cells produce IL‐10 and that exogenous IL‐10 causes downregulation of CD40 expression on DCs. In addition, endogenous IL‐10 produced by colon carcinoma cells inhibited CD40‐dependent IL‐12 production by DCs since tumor‐induced inhibition of IL‐12 production was abrogated by neutralizing anti‐IL‐10 antibody. Finally, systemic administration of FLT3L and/or CD40L reversed CD40 and IL‐12 (p40) deficiency of DCs in tumor‐bearing mice in vivo. These findings thus demonstrate that tumor‐derived factors, including IL‐10, inhibit CD40 expression on DCs and DC precursors and suppress their maturation and function.