Anat Cell Biol.  2013 Dec;46(4):262-271. 10.5115/acb.2013.46.4.262.

Suppression of in vitro murine T cell proliferation by human adipose tissue-derived mesenchymal stem cells is dependent mainly on cyclooxygenase-2 expression

• Kim JH
• Lee YT
• Hong JM
• Hwang YI

• KMID: 2046767
• DOI: http://doi.org/10.5115/acb.2013.46.4.262

Abstract

Mesenchymal stem cells (MSCs) of human origin have been frequently applied to experimental animal models to evaluate their immunomodulatory functions. MSCs are known to be activated by cytokines from T cells, predominantly by interferon-gamma (IFN-gamma), in conjunction with other cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interlukin-1beta. Because IFN-gamma is not cross-reactive between human and mouse species, the manner in which human MSCs administered in experimental animals are activated and stimulated to function has been questioned. In the present study, we established MSCs from human adipose tissue. They successfully suppressed the proliferation of not only human peripheral blood mononuclear cells but also mouse splenic T cells. When these human MSCs were stimulated with a culture supernatant of mouse T cells or recombinant murine TNF-alpha, they expressed cyclooxygenase-2 (COX-2), but not indoleamine 2,3-dioxygenase. The dominant role of COX-2 in suppressing mouse T cell proliferation was validated by the addition of COX-2 inhibitor in the co-culture, wherein the suppressed proliferation was almost completely recovered. In conclusion, human MSCs in a murine environment were activated, at least in part, by TNF-alpha and mainly used COX-2 as a tool for the suppression of in vitro T cell proliferation. These results should be considered when interpreting results for human MSCs in experimental animals.

Keyword

Mesenchymal stem cells; Humans; Cyclooxygenase-2; Immunomodulation

MeSH Terms

Adipose Tissue
Animals
Cell Proliferation*
Coculture Techniques
Cyclooxygenase 2*
Cytokines
Humans*
Immunomodulation
Indoleamine-Pyrrole 2,3,-Dioxygenase
Interferon-gamma
Mesenchymal Stromal Cells*
Mice
Models, Animal
T-Lymphocytes
Tumor Necrosis Factor-alpha
Cyclooxygenase 2
Cytokines
Indoleamine-Pyrrole 2,3,-Dioxygenase
Interferon-gamma
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Establishment of human mesenchymal stem cells (MSCs) from adipose tissue. MSCs were isolated from human fat tissue. (A) On day 3 of culture, cells adhered to the bottom of the culture dish and appeared spindle-shaped. On day 3, cells cultured in EMG-2 media were nearly 90% confluent (left panel), while cells in mixed media were about 50% confluent (right panel). (B) The obtained cells were stained with antibodies against various surface markers, as indicated, and analyzed by flow cytometric analysis. Cell populations were negative for hematopoietic markers (CD34, CD45, and CD117), and positive for stem cell markers (CD29, CD44, CD90, and CD105). (C) Cells were induced to differentiate to adipocytes (left panel), chondrocytes (middle panel), or osteoblasts (right panel) using corresponding differentiation medium, fixed with 4% paraformaldehyde at an appropriate time point, and stained with Oil Red-O, Alcian blue, or Alizarin red S, respectively.

• 

  Fig. 2 Suppressive effects of human adipose tissue-derived mesenchymal stem cells (hAd-MSCs) on the proliferation of T cells. Human peripheral blood mononuclear cells (PBMCs) (A), mouse splenocytes (B), and mouse splenic T cells (C) were stimulated with antibodies against human or mouse CD3 and CD28, respectively, and cultured in the presence or absence of hAd-MSCs (1:10 ratio of MSCs:target cells) for 2 days. 3[H]-thymidine was added to the culture, and radioactivity was determined after 18 hours. The presence of hAd-MSCs markedly suppressed T cell proliferation of both human and mouse cells. Results are representative of three independent experiments. CPM, count per minute. *P<0.001.

• 

  Fig. 3 Expression of immunomodulatory molecules in activated human adipose tissue-derived mesenchymal stem cells (hAd-MSCs). (A) Peripheral blood mononuclear cells (PBMCs) or mouse T cells were activated with concanavalin A (ConA), with antibodies against human or mouse CD3 and CD28, or with phorbol myristate acetate/ionomycin (PMA+I) for 3 days, and the supernatants were thus obtained. hAd-MSCs were stimulated with each supernatant for 24 hours and harvested. Total RNA was purified from these cells, and reverse transcription polymerase chain reaction analysis was performed for the molecules as indicated. (B) The average values of the intensities of the polymerase chain reaction bands from three replicate experiments measured by densitometry are shown. Lines at the top of each bar represent the magnitude of standard deviation. COX-2, cyclooxygenase-2; iNOS, inducible NO synthase; IDO, indoleamine 2,3-dioxygenase.

• 

  Fig. 4 Restoration of the suppressive effect of human adipose tissue-derived mesenchymal stem cells (hAd-MSCs) by the cyclooxygenase-2 (COX-2) inhibitor NS-398. (A) Mouse splenic T cells were co-cultured with hAd-MSCs in the presence or absence of 0.5 mM NS-398 (left panel), or in the presence or absence of 0.5 mM 1-MT (indoleamine 2,3-dioxygenase [IDO] inhibitor), or 1 mM L-NAME (right panel) for 2 days. 3[H]-thymidine was added to the co-culture, and radioactivity was determined after 18 hours. The presence of hAd-MSCs markedly suppressed the T cell proliferation of mouse T cells, which was almost completely recovered by the addition of the COX-2 inhibitor (left panel), while the addition of the IDO inhibitor or inducible NO synthase inhibitor did not affect the suppressive function of hAd-MSCs (right panel). (B) hAd-MSCs were stimulated with the culture supernatant of mouse T cells or with each recombinant mouse cytokine including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interlukin (IL)-4, or IL-5 at a concentration of 1 ng/ml. Cells were harvested after 24 hours, total RNA was extracted, and reverse transcription polymerase chain reaction was performed for COX-2 expression. Only the hAd-MSCs stimulated with culture supernatant or TNF-α expressed the molecule. Each experiment was repeated more than three times with representative results depicted here. CPM, count per minute; DMSO, dimethyl sulfoxide, vehicle control; Sup, culture supernatant of mouse T cells; *P<0.01.

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