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Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture
Published on: April 8, 2016
Cytokine pattern of Langerhans cells isolated from murine epidermal cell cultures
S Schreiber1, O Kilgus, E Payer
1Department of Dermatology I, University of Vienna Medical School, Austria.
Insights
Cultured Langerhans cells (cLC) primarily secrete IL-6 and IL-1 beta, crucial for T cell activation. Keratinocytes (KC) produce IL-1 alpha, GM-CSF, and TNF-alpha, highlighting distinct cytokine profiles.
Area of Science:
- Immunology
- Cell Biology
Background:
- Langerhans cells (LC) are key immune cells in the skin.
- Understanding LC cytokine production is vital for immunology research.
- Contamination with keratinocytes (KC) complicates LC cytokine analysis.
Purpose of the Study:
- To determine the specific cytokine secretion profile of cultured murine Langerhans cells (cLC).
- To differentiate cytokine production between cLC and contaminating keratinocytes (KC).
- To investigate the synthesis and release of cytokines by cLC and KC.
Main Methods:
- Supernatant cytokine bioactivity assays.
- Neutralizing antibody (mAb) blockade experiments.
- Polymerase chain reaction (PCR) and Northern blot analysis for cytokine transcripts.
- In situ hybridization for single-cell mRNA detection.
Main Results:
- cLC supernatants showed IL-1, IL-6, GM-CSF, and TNF-alpha activities, with IL-6 and IL-1 beta bioactivities blocked by specific mAbs.
- Cell mixing experiments indicated cLC are the major source of IL-6, while KC produce IL-1, GM-CSF, and TNF-alpha.
- Molecular studies confirmed cLC synthesize IL-1 beta and IL-6 transcripts, whereas KC produce IL-1 alpha and GM-CSF transcripts.
Conclusions:
- Cultured murine LC synthesize and secrete IL-1 beta and IL-6.
- KC are the primary producers of IL-1 alpha, GM-CSF, and TNF-alpha.
- IL-6 and IL-1 beta from cLC are important accessory molecules in T cell activation.
Abstract:
In the present study we demonstrate that supernatants of highly enriched cultured Langerhans cells (cLC) display IL-1, IL-6, granulocyte/macrophage (GM)-CSF, and TNF-alpha, but no IL-2, IL-3, IL-4, and IFN-gamma activities. We further show that IL-6, GM-CSF, and TNF-alpha bioactivities can be specifically blocked in the presence of the respective neutralizing mAb. Concerning the IL-1 bioactivity, the combined use of anti-IL-1 alpha and anti-IL-1 beta mAb was needed to completely inhibit the proliferative response of the indicator cell line D10. One of the difficulties in studying the secretory potential of LC is that even highly enriched cLC are contaminated with keratinocytes (KC), which are known to be a rich source of cytokines. To overcome this problem we compared cytokine bioactivities in supernatants of cell cultures consisting of selected cLC:cKC ratios. These cell mixing experiments revealed that cLC are the major source of the IL-6 bioactivity, whereas IL-1, GM-CSF, and TNF-alpha are predominantly generated by cKC. In order to determine whether the cytokine bioactivities measured in supernatants of epidermal cell cultures are simply caused by an increased release or by de novo synthesis, we performed molecular biologic studies. Polymerase chain reaction analysis of cLC and cKC revealed that IL-1 beta and IL-6 transcripts are virtually limited to cLC, whereas IL-1 alpha, GM-CSF, and TNF-alpha messages are preferentially exhibited by cKC. mRNA coding for IL-2, IL-3, IL-4, and IFN-gamma could neither be amplified from cLC nor from cKC. Furthermore, the quantitative comparison of cytokine transcripts in cLC vs cKC using Northern blot analysis and mRNA detection on the single cell level using in situ hybridization confirmed that cLC generate IL-6, whereas cKC synthesize IL-1 alpha and GM-CSF. Taken together our results demonstrate that cultured murine LC synthesize and secrete IL-1 beta and IL-6, cytokines known to be important accessory molecules in T cell activation.
