Attraction Of Immune Cells Research Articles

Cleavage of sphingosine kinase 2 by caspase-1 provokes its release from apoptotic cells

AbstractExecution of physiologic cell death known as apoptosis is tightly regulated and transfers immunologically relevant information. This ensures efficient clearance of dying cells and shapes the phenotype of their “captors” toward anti-inflammatory. Here, we identify a mechanism of sphingosine-1-phosphate production by apoptotic cells. During cell death, sphingosine kinase 2 (SphK2) is cleaved at its N-terminus in a caspase-1–dependent manner. Thereupon, a truncated but enzymatically active fragment of SphK2 is released from cells. This step is coupled to phosphatidylserine exposure, which is a hallmark of apoptosis and a crucial signal for phagocyte/apoptotic cell interaction. Our data link signaling events during apoptosis to the extracellular production of a lipid mediator that affects immune cell attraction and activation.

Substance P alters the in vitro LPS responsiveness of bovine monocytes and blood-derived macrophages

Neuromediators like substance P have a decisive influence on inflammatory processes via the neuroendocrine regulation circuit. The aim of the present study was therefore to evaluate the expression of the main substance P receptor NK-1R in cattle as well as the modulatory properties of substance P for bovine macrophages. The expression of NK-1R was detected in subsets of lymphocytes, granulocytes, monocytes and in vitro-generated macrophages (MdM). Stimulation of monocytes and MdM with lipopolysaccharide (LPS) for 3 h did not alter the expression level of NK-1R. In vitro, the modulatory potential of substance P for monocytes and in vitro-generated blood-derived macrophages (MdM) was analysed. In MdM, generated in the presence of substance P, mRNA expression of chemokines, which are crucial for the attraction and activation of granulocytes and monocytes (CXCL8, CCL5) as well as the expression of IL-1β, a classically pro-inflammatory cytokine were significantly elevated. After stimulation with LPS, MdM generated in the presence of substance P showed an elevated expression of CXCL8 and IL-1β, while in SP-influenced monocytes only the expression of CCL5 was significantly upregulated after LPS stimulation. In addition, supernatants of MdM cultured in the presence of substance P induced neutrophil migration and inhibited both necrosis and apoptosis of neutrophil granulocytes. Thus, it has been shown that the modulation of the expression pattern of chemokines and cytokines in MdM by substance P has also functional relevance for the attraction and activation of other immune cells. In general, the modulation of sensor and effector functions by substance P suggests, that this neuromediator can alter the course of an inflammatory disease in cattle.

Differential expression of the chemokines GRO-2, GRO-3, and interleukin-8 in colon cancer and their impact on metastatic disease and survival

Chemotactic cytokines play a role in angiogenesis and attraction of immune cells. However, their contribution to tumor formation remains incompletely understood. In a previous transcriptome study, we identified a family of structurally related chemokines of the CXC-family to be specifically up-regulated in colorectal cancer. The aim of the present study was to investigate the regulation of their expression in colon cancer cells and to test the hypothesis that altered CXC-chemokine expression is related to critical clinical parameters, such as survival or metastasis formation. Expression levels of interleukin-8 (CXCL-8) and growth-related oncogenes 2 and 3 (GRO-2/CXCL-2 and GRO-3/CXCL-3) were quantified using qRT-PCR in 97 patients with completely resected colon carcinoma and correlated with clinical parameters. Moreover, 16 samples of normal mucosa, nine samples of benign adenoma, and 11 samples of liver metastasis were analyzed. Next, the regulation of chemokine expression in response to various stimuli was tested in colon cancer cell lines (HT29, HCT116, CaCO2). Expression of GRO-2, GRO-3, and IL-8 was significantly increased in colon cancer as compared to normal colon tissue. Expression of GRO-2 and GRO-3 was already enhanced in premalignant adenomas, and GRO-3 was significantly down-regulated in liver metastasis as compared to the primary tumor. Importantly, expression of GRO-3 was significantly higher in patients with local versus systemic disease. Moreover, IL-8 expression was significantly associated to overall post-operative survival. Finally, all chemokines were strongly induced by IL-1alpha in the colon cancer cell lines tested, indicating a potential link to inflammatory processes. In accordance with earlier findings, we report here a significantly increased expression of GRO-2, GRO-3, and IL-8 in colon carcinoma as compared to normal tissue. Furthermore, GRO-3 was related to metastasis formation, and IL-8 was associated with survival, suggesting a potential predictive power of these markers.

Role of Senescence in Dynamic Tumor/Host Responses to Cancer Therapy In Vivo.

Abstract 928 Introduction:Premature senescence is a terminal G1 growth arrest in response to acute cellular stresses, such as oncogene activation or exposure to DNA damaging chemotherapy, with tumor-suppressive activity in vivo. However, senescent cells remain viable. They are metabolically active and have a distinct senescence-associated secretory profile (SASP). Therefore, the function and the fate of senescent cells within the tumor site, particularly their impact on the tumor microenvironment, are highly complex and not well characterized. Here, we analyze lymphoma cells with defined genetic lesions, e.g. deletion of the histone H3 lysine 9 methyltransferase Suv39h1 (controlling senescence) and p53 (mediating both apoptosis and senescence), for their influence on immunological tumor-host and growth-modulating senescent/non-senescent cell interactions as a consequence of therapy-induced senescence (TIS) in the Eμ-myc mouse lymphoma model. We identify novel cell-autonomous and non-cell-autonomous components of the senescence response in vitro and in vivo, which arise as new targets for lymphoma therapy. Methods: Lymphoma cells (LCs) from different genetic backgrounds (Suv39h1-, p53null, atm-/-, p16INK4a-/-, p19ARF-/-, p21CIP1-/-) were retrovirally transduced with the bcl2 gene to block apoptosis. Subsequently, they were treated with the DNA damaging anticancer agent adriamycin in vitro or the alkylating agent cyclophosphamide upon lymphoma formation in normal immunocompetent mice in vivo. TIS was detected based on senescence-associated β-galactosidase activity (SA-β-gal), Ki67 staining and BrdU incorporation. The SASP of senescent LCs was analysed by gene expression and cytokine arrays. A large pharmacological inhibitor screen was used to identify signalling cascades activated by the SASP. To study tumor-host cell interactions in vitro, freshly isolated spleen cells were co-incubated with proliferating or TIS LCs. Immunophenotyping was carried out with leukocyte-specific antibodies. Immune responses elicited upon TIS induction in vivo were further analysed in gld (generalized lymphoproliferative disease) mice, which lack functional FasL, and by systemic depletion of macrophages after clodronate administration. Results:TIS lymphoma cells, but not Suv39h1- or p53-deficient LCs, exhibit a SASP with pro-inflammatory and pro-senescent action on tumor and bystander cells. We identify novel components of the SASP profile with the potential to induce a secondary senescent arrest in bcl2 protected Eμ-myc lymphoma cells via the MAPK and TGFβ pathway. In vivo, TIS correlates with the attraction of immune cells to the tumor site and subsequent clearing of senescent cells, which can be attenuated by systemic depletion of macrophages and interference with T cell- mediated programmed cell death. Senescent LCs interact with different immune cell subsets in vitro, in particular macrophages, granulocytes and T-cells, and become sensitive to macrophage engulfment and death-receptor mediated apoptosis, for example by Fas-FasL cytotoxicity. Discussion:This study demonstrates that therapy-induced senescence drives a profound remodeling of the tumor site after therapy and unveils functional interactions of senescent LCs with proliferating tumor cells and different immune cell subsets in vitro and in vivo. Senescent cells secrete a cytokine program, which limits tumor growth and stimulates immune cell attraction, hereby promoting their own clearance. Thus, TIS is a highly dynamic and interdependent process whose paracrine effects and immune cell interactions account for regression of the senescent mass and present an attractive target network for novel therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Cytokines and chemokines in testicular inflammation: A brief review

A wide spectrum of data in the literature shows the relevance of cytokines as paracrine regulators of spermatogenesis and steroidogenesis in the normal testis. In this brief review, we highlight the relevance of cytokines in the testis during inflammation. This phenomenon involves complex and multiple interactions among immune and germ cells generally resulting in the alteration of spermatogenesis. The complexity of these cell interactions is multiplied because Sertoli and Leydig cells are also producers of pro- and anti-inflammatory cytokines and chemokines. Also, cytokines are pleiotropic and they exert opposite and/or redundant effects in different conditions. However, in spite of this bidirectional immunoregulatory function of cytokines, the mass of the data, reported from experiments of acute testicular inflammation, shows upregulation of interleukin (IL)-1beta, IL-1alpha, IL-6, and tumor necrosis factor alpha (TNF-alpha), which induce adverse effects on germ cells. In autoimmune orchitis, a chronic testicular inflammation, chemokines such as CCL2, CCL3, and CCL4 induce attraction and extravasation of immune cells within the testicular interstitium. These cells alter the normal immunosuppressor microenvironment principally through the secretion of proinflammatory cytokines, interferon-gamma initially, and IL-6 and TNF-alpha thereafter. Germ cells expressing TNFR1, IL-6R, and Fas increase in number and undergo apoptosis, through the TNF-alpha/TNFR1, IL-6/IL-6R, and Fas/Fas L systems. The knowledge of immune-germ and somatic testicular cell interactions will contribute to the understanding of the mechanisms by which chronic inflammatory conditions of the testis can disrupt the process of spermatogenesis.

Cellular Senescence Contributes to the Outcome of Lymphoma Chemotherapy by Cell-Autonomous and Paracrine Immune Mechanisms

Introduction: Premature senescence reflects an acutely inducible, irreversible growth arrest as a cellular response to stresses such as oncogenic activation and DNA damage, including chemotherapeutic anticancer agents. Senescence complements apoptosis as a tumor suppressive and therapeutic effector principle, but whether a selective disruption of the senescence machinery impairs treatment outcome is unknown. Moreover, function and fate of senescent tumor cells within the tumor site remain unclear. Here, we analyze the impact of defined genetic alterations, i.e. Bcl2 overexpression (blocking apoptosis), deletion of the histone H3 lysine 9 methyltransferase Suv39h1 (controlling senescence), and conditional expression of p53 (mediating both apoptosis and senescence), on therapy-induced senescence (TIS) in the Eμ-myc mouse lymphoma model with specific emphasis on immunological tumor-host and growth-modulating senescent/non-senescent cell interactions as a consequence of TIS in vitro and in vivo.Methods: Lymphoma cells (LCs) of various genetic backgrounds were retrovirally transduced with the bcl2 gene to study TIS in the absence of drug-induced apoptosis. Bcl2-protected LCs were treated with the DNA damaging anticancer agent adriamycin in vitro, or were exposed to the alkylating agent cyclophosphamide upon lymphoma formation in normal immunocompetent mice in vivo. TIS was detected by staining for senescence-associated β-galactosidase activity (SA-β-gal) and other senescence-related markers, including Ki67 and BrdU incorporation. To study tumor-host cell interactions, isolated normal splenocytes were co-incubated with proliferating or senescent LCs in vitro. Immunophenotyping was carried out with antibodies specific for macrophages, granulocytes, natural killer cells and T-lymphocytes. Cytokine production was measured by protein arrays.Results: Senescent LCs engage in cell-cell interactions with different immune cell subsets, in particular macrophages, granulocytes and T-cells in vitro. Fluorescence microscopy reveals that macrophages engulf LCs after they entered TIS. In vivo, TIS correlates with the quantitative attraction of immune cell populations to the tumor site and subsequent clearing of senescent cells. Ongoing mechanistic studies on underlying ligand/receptor interactions will be reported at the meeting. TIS cells exhibit a specific pro-inflammatory secretory profile whose functional impact on tumor and bystander cells is currently being investigated. Importantly, this profile is distinguishable from cytokine profiles of senescence-compromised Suv39h1- or p53-deficient lymphomas, and, thus, reflects a senescence - rather than a DNA damage-associated secretory response.Discussion: The study unveils a functional interaction of senescent LCs with different immune cell subsets in vitro and in vivo. The cytokine arrays show that senescent cells produce a specific secretory profile, which might stimulate immune cell attraction. Therefore, immune cells could be recruited to lymphomas in vivo specifically after TIS with the potential to clear senescent – and possibly non-senescent – cells from the tumor site. The data demonstrate genetically that senescence is a beneficial effector principle of DNA damaging chemotherapy and encourage further exploration of this program to limit cancer expansion in vivo.

Molecular profiling reveals distinct functional attributes of CD1d-restricted natural killer (NK) T cell subsets

CD1d-restricted natural killer T (NKT) cells can have multiple effects on an immune response, including the activation, regulation and attraction of innate immune cells, and modulation of adaptive immunity. Recent studies reveal that there are distinct subsets of NKT cells which selectively perform some of the functions attributed to CD1d-restricted cells, but the mechanisms underlying these functional differences have not been resolved. Our aim in this study was to identify novel NKT cell associated traits that would provide important insight into NKT cell activation and function. To this end, we have performed gene expression profiling of two separate subsets of NKT cells, analyzing genes differentially expressed in these cells compared to conventional CD4 +NK1.1 − T cells. We identify different sets of genes over expressed in each of the two NKT cell types, as well as genes that are common to the two CD1d-restricted NKT cell populations analyzed. A large number of these genes are highly relevant for NKT cell development, activation and function. Each NKT subtype displayed a unique set of chemokine receptors, integrins and molecules related to effector function, supporting the notion that distinct NKT cells can be selectively engaged and have diverse functions in different types of immune reactions.

NOD β-cells express RANTES and may attract their own killers

In autoimmune diabetes, the phase of the T cell dependent destruction of the insulin-producing β-cells of the pancreas is preceded by the infiltration of the islets of Langerhans with immune cells (insulitis). The mechanisms underlying this process are poorly understood. However, chemokines, a family of proteins known to be key players in the regulation of lymphocyte trafficking, are thought to be key players in regulating the directed attraction of immune cells to the islets of Langerhans. Recently, it has been demonstrated that pathogenic T cells bear the chemokine receptor CCR4. CCR4 positive cells cause very efficiently type-1-diabetes upon transfer and expand in secondary lymphoid organs during progression to overt diabetes. However, it remains enigmatic of how these T cells are recruited from secondary lymphoid organs to the islets of Langerhans. The chemokines MDC, one ligand of CCR4, could not be detected within the islets. As TARC, another CCR4 ligand, is expressed at low levels by some infiltrating cells, but not by islet cells, TARC expression may be involved in the pathogenic process once insulitis is manifest but can’t be responsible for the initial recruitment of T cells to the islets. Here, applying classical immunhistochemistry and single cell immunofluorescense, we report for the first time that NOD β-cells express RANTES, the third ligand of CCR4. Therefore, β-cells may be responsible for the initial attraction of their own killers, the CCR4-bearing diabetogenic T cells. Moreover, given that RANTES is known to be able to activate T cells and to enhance CTL cytotoxicity, β-cell-derived RANTES may have a major impact on the pathogenic microenvironment within the islet, promoting the autoimmune process. Finally, RANTES expression by β-cells may constitute a key element in an amplificatory loop: Once T cells have reached the pancreas, cytokines secreted by these T cells may augment RANTES expression by β-cells which in turn causes further attraction and activation of immune cells. Targeting the action of β-cell-derived RANTES may be, therefore, a promising therapeutic approach to prevent autoimmune diabetes.

Chimiokines et pathologie allergique

Chemokines are small proteins responsible for the attraction of immune cells to sites of inflammation. They therefore play a central role in the inflammatory response, and the type of reaction depends on the chemokines involved. Fifty chemokines have been identified to date. Their receptors on the target cell surface are varied; 19 of these have now been identified. Some of them can link one chemokine, and vice versa, meaning that the chemokine network is complex. Nevertheless, some receptors and some chemokines appear to be relatively specific for a given cell type or for a group of cells involved in the same kind of reaction. With regard to allergy, the chemokines CCL-5 (RANTES), CCL-11 (Eotaxin 1), and CCL-7 (MCP-3) and their common receptor CCR-3 seem particularly involved. These chemokines and this receptor therefore appear to be potentially important targets for future anti-allergic treatments.

Combination Gene Therapy of Herpes Simplex Virus Thymidine Kinase and Cytokines in Lung Cancer

Background : One of the important mechanisms responsible for a tumor escaping the immune response is an absence of the tumor associated antigen (TAA) on the cancer cell surface. To overcome this, combination gene therapy using a herpes simplex thymidine kinase (HSTK) gene, prototype of drug sensitizing gene, was conducted to enhance T AA release by cell destruction, as well as the cytokine genes for immune cell attraction. Methods : We investigated whether or not transduction with the adenovirus-HSTK (Ad-HSTK) enhanced the sensitivity of Lewis lung carcinoma (LLC) to ganciclovir (GCV) and induced a bystander effect. A Tumor vaccine trial was performed using LLC with ad-HSTKad-GM-CSFad-IL-2 to determine if they exhibit some antitumor effect on established lung cancer xenografts. Results : LLC with ad-HSTK revealed a much higher sensitivity to ganciclovir (GCV). LLC transduced with ad-HSTK and/or ad-IL-2, ad-GM-CSF showed a lower in vivo tumorigenicity. In the treatment experiment, vaccination with LLC transduced with ad-HSTK, ad-IL-2, or ad-GM-CSF alone modestly suppressed the growth of an established tumor. Combined transduction with HSTK and GM-CSF induced stronger growth suppression of a established lung cancer, while HSTK and IL-2 combination transduction did not have any antitumor effect on individual transduction. Vaccination with LLC-HSTK-GM-CSF increased the infiltration of dendritic cells in the spleen. Conclusion : It was concluded that a tumor vaccine transduced with HSTK and GM-CSF induces strong antitumor immunity by activating the dendritic cells.

Different sensitivities of the murine melanomas BL-6 and BL-6-??m to local injections of interleukin-2 (IL-2). Analysis of gangliosides after the treatment

The murine melanoma cell line BL-6-beta m, which is a stable cell line transfected with a gene coding a unique actin subspecies called beta m to the BL-6 cell line, has low metastatic potentials as compared with those of the parent cell line. BL-6-beta m melanomas were found to be sensitive to in vivo local injection of IL-2, while BL-6 melanomas showed almost no response. Ganglioside analysis of BL-6 and BL-6-beta melanomas revealed that the main ganglioside of both melanomas was GM3, which suggested that different sensitivities between BL-6 and BL-6-beta m melanomas to the injection of IL-2 did not relate to the different compositions of main gangliosides. However, minor components of the gangliosides such as GM2 and GM1 emerged only in BL-6-beta m melanomas after treatment with IL-2. Local injection of IL-2 caused considerable infiltration of anti-asialo GM1-positive cells into the nests as well as the interstitials of BL-6-beta m melanomas. In contrast, in the BL-6 melanomas treated with IL-2, infiltration of the anti-asialo GM1-positive cells was hardly seen, although anti-Thy1,2 and anti-macrophage-positive cells were found to more or less the same extent as observed in BL-6-beta m melanomas. These results suggest that the murine metastatic variant melanoma cell lines BL-6 and BL-6-beta m have different properties in terms of sensitivity to in vivo IL-2 treatment, and a slight enhancement of the ganglioside components GM2 and GM1 expression only in BL-6-beta m after IL-2 treatment may play a role in the IL-2-mediated attraction of immune cells or may explain the different sensitivities of the two lines to treatment with IL-2.