Elsevier

Cytokine

Volume 25, Issue 1, 7 January 2004, Pages 31-35
Cytokine

Cytokine and chemokine expression profiles of maturing dendritic cells using multiprotein platform arrays

https://doi.org/10.1016/j.cyto.2003.08.012Get rights and content

Abstract

Understanding the whole process of dendritic cell (DC) activation might help in the development of more efficient immunotherapeutic strategies for tumor patients. Part of this process is cytokine secretion, which has important effects on innate and adaptive immune response. Here, we cultured circulating monocytes for five days with interleukin-4 and GM-CSF followed by two-day culture with or without CD40 ligand and LPS to create a mature DC (mDC) and an immature DC (iDC) phenotype, respectively, characterized by differential expression of co-stimulatory molecules (CD80, CD83). We then compared the cytokine expression profile of the mDC and iDC using two protein platform arrays. Twelve supernatants from mDC paired with 12 from iDC were compared. The mDC protein expression profile showed significant increases in 16 out of 34 factors tested, including TNFα, IL-10, IL-12, IFNγ, MIP1α, MIP1β, IL-8, MDC, RANTES, and IL-6, which play a crucial role in the regulation of the innate immune response as well as the recruitment and activation of adaptive immune effectors. Interestingly, some of the cytokines expressed during maturation were also found in the gene expression profile identified in tumor metastases following IL-2 therapy using cDNA arrays. This finding suggests a possible role for resident DC maturation as a mediator of systemic IL-2 effects. Most important, the array of cytokines secreted during DC maturation may be considered an important component during adoptive transfer. Further characterization of the kinetics and persistence of their secretion should be undertaken in the future.

Introduction

Understanding the process of dendritic cell (DC) activation might be a key to the development of more efficient immunotherapeutic strategies for treating patients with cancer [1]. In the last decade, interest in the biology of DC resident in tumor deposits or adoptively transferred for vaccination purposes has consistently increased. Resident DC might influence the development of a natural immune response and mediate the intensity of therapy-induced immune responses. In previous research, we studied the gene expression profile of metastatic deposits in patients with melanoma undergoing high-dose interleukin-2 (IL-2) therapy using an 8000-gene cDNA microarray [2]. Comparative analysis of the transcriptional changes occurring at tumor sites and in peripheral blood mononuclear cells (PBMC) pre- and post-IL-2 administration demonstrated predominant up-regulation of DC-associated cytokines, chemokines, and respective receptors, suggesting their central role in the target organ during systemic IL-2 therapy.

DC also offer great potential in the context of active-specific vaccination against cancer [1], [3]. Several early-phase clinical studies show effectiveness of DC-based vaccination [4], [5]. Most studies suggest that DC maturation is a requirement for the development and maintenance of tumor-directed T cell responses [6], [7], [8]. While immature DC (iDC) induce regulatory, IL-10-producing CD4+ and CD8+ T cells, mature DC (mDC) induce functionally active IFN-γ-producing CD8+ T cells [1]. There is also preliminary evidence that mDC are able to induce stronger T cell responses than iDC in tumor patients [9].

Gene expression profiling of DC during different maturation steps identified the transcription of various cytokines and chemokines including IL-10, IL-12, IL-6, TNFα, TARC, RANTES, MCP-1, CCR7, MIP-3β, and MIG [10], [11], [12]. These results open the question of whether a corresponding gene product could be observed in similar conditions. However, to our knowledge no comprehensive protein-based investigation of the secretory response of DC in response to maturation stimuli has been reported. Secretion of a few cytokines during DC activation has been characterized including IL-6, IL-10, IL-12, IL-2, and TNFα [11], [13], [14], [15]. In addition, using a traditional ELISA, MIP-1α, MIP-1β, and IL-8 were observed in response to maturation stimuli [16]. Since cytokines might serve an important role in linking innate and adaptive anti-tumor-antigen immune responses [17], we performed this explorative study to identify the most consistently expressed immune mediators that accompany DC maturation. Innovation in high throughput proteomic platforms allows the analysis of a large number of molecules in one setting using relatively small amounts of material. Using such tools, we compared the cumulative secretion of cytokines during 48 h of DC maturation with CD40L and LPS using two multiprotein platform arrays.

Section snippets

Maturation of DC

Phenotypic analyses of mDC and iDC consistently demonstrated that HLA-DR antigens and CD86 were expressed at high levels, while mature DC expressed consistently and significantly more CD80 and CD83 than iDC. Thus, the present treatment with CD40L and LPS generated two subsets of DC in distinct stages of activation. A representative example is given in Fig. 1.

Differential cytokine/chemokine expression by iDC and mDC

IL-4 and GM-CSF were highly expressed in both mature and immature DC supernatants due to the fact that they had been added for generation

Discussion

This work confirms previous reports that documented the release of several cytokines and chemokines during DC maturation and at the same time broadens the information to a larger set of putative immune effectors [11], [13], [14], [16]. Thus, in addition to the up-regulation of co-stimulatory molecules, cytokine release is a major component of DC activation. This is not surprising as cytokines and chemokines may play an important role in activating effectors of the innate immune response, which

Generation and maturation of dendritic cells and collection of supernatants

PBMC were collected from eight healthy donors (one donor: three samples, three donors: two samples, and four donors: one sample) in the Department of Transfusion Medicine, Clinical Center, Bethesda, MD. All cells were maintained in complete medium (CM) consisting of Iscove's medium (Biofluids, Rockville, MD) supplemented with 10% heat-inactivated human AB-serum (Gemini Bioproducts, Inc, Calabasas, CA), 10 mM hepes buffer (Cellgro, Mediatech, Inc. Herndon, VA), 0.03% l-glutamine (Biofluids), 100

Acknowledgements

Dirk Nagorsen is supported by the Dr. Mildred Scheel Stiftung für Krebsforschung (Deutsche Krebshilfe), Home institution: Med. Klinik III, Hematology, Oncology, and Transfusion Medicine, UKBF, FU Berlin, Germany.

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