Cytokine and adhesion molecule expression in primary human endothelial cells stimulated with fever-range hyperthermia.

Migration of blood-borne lymphocytes into lymphoid tissues and sites of inflammation is initiated by vascular adhesion molecules and proinflammatory cytokines. Previous in vivo studies have shown that febrile temperatures dynamically stimulate adhesion in differentiated high endothelial venules (HEV), which are portals for lymphocyte extravasation. This report examines the direct effect of fever-range hyperthermia on the expression of adhesion molecules and cytokines by primary cultured endothelial cells. In both macrovascular (HUVEC) and microvascular (HMVEC) endothelial cells, fever-range hyperthermia (40 degrees C for 6-12 h) did not affect expression of adhesion molecules (ICAM-1, E-selectin, VCAM-1, P-selectin, PECAM-1, PNAd, MAdCAM-1), cytokine release (IL-1beta, TNF-alpha, IFN-gamma, IL-6, IL-11, IL-12, IL-13), or chemokine secretion (IL-8, RANTES, MCP-1, MIP-1beta, MIG). This is in contrast to the stimulatory effects of TNF-alpha or 43 degrees C heat shock. However, a novel role for fever-range hyperthermia was identified in augmenting actin polymerization in cultured endothelial cells and enhancing the ability of endothelial-derived factors to transactivate the alpha4beta7 integrin lymphocyte homing receptor. These findings provide insight into the tightly regulated effects of fever-range hyperthermia that exclude induction of adhesion in non-activated endothelium of normal blood vessels. Through these mechanisms, it is proposed that febrile temperatures associated with infection or clinical hyperthermia avoid the unproductive exodus of lymphocytes to non-involved extralymphoid tissues while simultaneously promoting lymphocyte delivery to sites of immune activation.     

Fever-range hyperthermia stimulates alpha4beta7 integrin-dependent lymphocyte-endothelial adhesion

Migration of blood-borne lymphocytes into lymphoid tissues is initiated by the Lselectin and alpha4beta7 integrin adhesion molecules. Previous studies have shown that L-selectin adhesion is dynamically regulated by febrile temperatures. It is now reported that fever-range hyperthermia also acts directly on lymphocytes to enhance selected adhesive functions of alpha4beta7 integrin. Fever-range hyperthermia treatment in vitro (40^oC, 12h) of murine TK1 lymphoma cells and human peripheral blood lymphocytes (PBL) stimulates alpha4beta7 integrin-dependent adhesion to high endothelial venules (HEV) in Peyer's patch and mesenteric lymph node frozen sections. TK1 cells are alpha4beta7^hi L-selectin^lo, allowing for the analysis of alpha4beta7 integrin without contributions from L-selectin. Adhesion was further shown to involve alpha4beta7 integrin and its endothelial counter-receptor, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) using function-blocking antibodies (i.e. DATK32, HP2/1, MECA-367). Fever-range hyperthermia also promotes alpha4beta7 integrin-mediated aggregation of TK1 cells. In sharp contrast, hyperthermia fails to increase alpha4beta7 integrin adhesion to fibronectin by TK1 cells. Expression of the alpha4beta7 heterodimer on TK1 cells or human PBL is not altered by hyperthermia, suggesting that hyperthermia stimulates adhesion by enhancing alpha4beta7 integrin avidity rather than its cell surface density. These results provide a mechanism whereby febrile temperatures during infection or clinical hyperthermia potentially amplify the immune response by stimulating L-selectin and alpha4beta7 integrin-dependent homing of immune effector cells to lymphoid tissues.     

Fever-range hyperthermia stimulates alpha4beta7 integrin-dependent lymphocyte-endothelial adhesion.

Migration of blood-borne lymphocytes into lymphoid tissues is initiated by the L-selectin and alpha4beta7 integrin adhesion molecules. Previous studies have shown that L-selectin adhesion is dynamically regulated by febrile temperatures. It is now reported that fever-range hyperthermia also acts directly on lymphocytes to enhance selected adhesive functions of alpha4beta7 integrin. Fever-range hyperthermia treatment in vitro (40 degrees C, 12 h) of murine TK1 lymphoma cells and human peripheral blood lymphocytes (PBL) stimulates alpha4beta7 integrin-dependent adhesion to high endothelial venules (HEV) in Peyer's patch and mesenteric lymph node frozen sections. TK1 cells are alpha4beta7hi L-selectin(lo), allowing for the analysis of alpha4beta7 integrin without contributions from L-selectin. Adhesion was further shown to involve alpha4beta7 integrin and its endothelial counter-receptor, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) using function-blocking antibodies (i.e. DATK32, HP2/1, MECA-367). Fever-range hyperthermia also promotes alpha4beta7 integrin-mediated aggregation of TK1 cells. In sharp contrast, hyperthermia fails to increase alpha4beta7 integrin adhesion to fibronectin by TK1 cells. Expression of the alpha4beta7 heterodimer on TK1 cells or human PBL is not altered by hyperthermia, suggesting that hyperthermia stimulates adhesion by enhancing alpha4beta7 integrin avidity rather than its cell surface density. These results provide a mechanism whereby febrile temperatures during infection or clinical hyperthermia potentially amplify the immune response by stimulating L-selectin and alpha4beta7 integrin-dependent homing of immune effector cells to lymphoid tissues.     

In vitro models of lymphocyte transendothelial migration.

The processes of lymphocyte-endothelial cell interaction and the in vitro assays employed in their study are the subjects of this review. In motility assays in porous filters and gel matrices, it has been shown that lymphocyte migration can be modulated by interleukin-2 (IL-2), IL-3, IL-4, IL-6, and IL-8. Cytokines can also modulate lymphocyte-endothelial adhesion. Endothelial intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) are induced or upregulated by IL-1 or tumor necrosis factor. In addition, interferon-gamma upregulates ICAM-1, and IL-4 can induce VCAM-1. The roles of these cytokines and adhesion molecules in transendothelial migration may be studied in assays in which lymphocytes penetrate layers of cultured endothelial cells. These models can distinguish lymphocyte adhesion from subsequent migration. Using such models, we and others have obtained evidence that both lymphocyte function-associated antigen-1 (LFA-1)/ICAM-1 and very late activation antigen 4 (VLA-4)/VCAM-1 interactions mediate lymphocyte adhesion to endothelial cells, but that LFA-1/ICAM-1 interactions play a greater role in transendothelial migration.     

Targeted regulation of a lymphocyte-endothelial-interleukin-6 axis by thermal stress

Immune protection from microbial invaders or malignant progression is dependent on the ability of lymphocytes to efficiently traffic across morphologically and biochemically distinct vascular sites throughout the body. Lymphocyte trafficking to target tissues is orchestrated by adhesion molecules and chemokines that stabilize dynamic interactions between circulating lymphocytes and endothelial cells lining blood vessels. While the molecular mechanisms that regulate the efficient migration of lymphocytes across specialized high endothelial venules (HEVs) in secondary lymphoid organs have been extensively characterized, there is a paucity of information available regarding the mechanisms that dictate the rate of lymphocyte entry into tumor tissues. This article summarizes recent evidence that inflammatory cues associated with fever-range thermal stress promote lymphocyte extravasation across HEVs of lymphoid organs through a highly regulated lymphocyte–endothelial–interleukin-6 (IL-6) biological axis. The potential for using thermally-based strategies to improve lymphocyte delivery to the tumor microenvironment during T cell-based immunotherapy will also be discussed.     

Targeted regulation of a lymphocyte-endothelial-interleukin-6 axis by thermal stress.

Immune protection from microbial invaders or malignant progression is dependent on the ability of lymphocytes to efficiently traffic across morphologically and biochemically distinct vascular sites throughout the body. Lymphocyte trafficking to target tissues is orchestrated by adhesion molecules and chemokines that stabilize dynamic interactions between circulating lymphocytes and endothelial cells lining blood vessels. While the molecular mechanisms that regulate the efficient migration of lymphocytes across specialized high endothelial venules (HEVs) in secondary lymphoid organs have been extensively characterized, there is a paucity of information available regarding the mechanisms that dictate the rate of lymphocyte entry into tumor tissues. This article summarizes recent evidence that inflammatory cues associated with fever-range thermal stress promote lymphocyte extravasation across HEVs of lymphoid organs through a highly regulated lymphocyte-endothelial-interleukin-6 (IL-6) biological axis. The potential for using thermally-based strategies to improve lymphocyte delivery to the tumor microenvironment during T cell-based immunotherapy will also be discussed.     

Role of carcinoembryonic antigen in the progression of colon cancer cells that express carbohydrate antigen

Carcinoembryonic antigen (CEA) has been reported to promote the metastatic potential in some experimental tumors. Adhesion molecules are known to play an important role in the process of metastasis. Cytokines, including interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), which are produced by Kupffer cells, induce endothelial cells to express adhesion molecules. As a result, the present study was designed to investigate whether the interaction between CEA and Kupffer cells accelerated the metastatic potential of tumors in the liver. Kupffer cells isolated from the liver of male BALB/c mice were cultured with CEA, either with or without the addition of a cytokine inhibitor. The levels of IL-1beta and TNF-alpha were examined in a culture medium. An adhesion assay of colon cancer cell lines to human umbilical vein endothelial cells was also performed. When CEA was added to the Kupffer cell culture medium, cytokines were produced. Elevated levels of cytokines appeared to lead to increased rates of adhesion of cancer cells to endothelial cells. However, these phenomena were blocked by the addition of cytokine inhibitors. CEA stimulated Kupffer cells to produce cytokines. An elevated number of cytokines have been proven to promote the expression of adhesion molecules in endothelial cells. These processes are therefore considered to contribute to the metastasis of malignant cells to the liver. These results suggest that cytokine inhibitors may therefore play an important role in the inhibition of hepatic metastasis.     

Importance of E-selectin (ELAM-1) and sialyl lewisa in the adhesion of pancreatic carcinoma cells to acttvated endothelium

Adhesion molecules involved in attachment between human pancreatic carcinoma and activated endothelial cells in vitro were investigated. Basal adhesion occurred between 6 pancreatic carcinoma cell lines and unstimulated human umbilical vein endothelial cells (HUVEC), and augmented basal adhesion to activated HUVEC was only seen when pancreatic cancer cells expressed sialyl Lewis^a (SLe^a) and sialyl Lewis^x (SLe^x). Activation of HUVEC with interleukin l-β (IL-Iβ) or tumor necrosis factor-α (TNF-α), but not with interferon-γ (IFN-γ), generated the augmentative basal adhesion. Dose dependence and additive effect were observed in augmentation of the basal adhesion induced by IL-I β and/or TNF-α. Increase in adhesion correlated with up-regulation of the surface E-selectin (or ELAM-I) on HUVEC, and was evident at both 25°C and 4°C. Anti-E-selectin and anti-SLe^a blocked the augmented attachment, whereas anti-SLe^x, an antibody against another known ligand for E-selectin, did not. The collective evidence indicates that attachment between pancreas carcinoma cells and activated endothelial cells is regulated by cytokines such as IL-1 β and TNF-α, and is mediated by SLe^a on pancreas carcinoma and E-selectin on endothelial cells. These molecules may be of significant importance in blood-borne metastasis of pancreatic carcinoma cells to inflamed sites.     

Artificial cytokine storm combined with hyperthermia induces significant anti-tumor effect in mice inoculated with Lewis lung carcinoma and B16 melanoma cells

In cancer immunotherapies combined with hyperthermia, one or two cytokines have been tested to augment the anti-tumor effect. However, the therapies have not shown sufficient improvement. The aim of this study is to find a new potent tumor immunotherapy in order to augment antitumor effect of hyperthermia by the cytokine cocktails in vivo. We used a combination therapy of local hyperthermia (LH) and various cytokine cocktails composed of IFNs (IFN-α, -β, and -γ), Th1 cytokines (IL-2, -12, -15, and -18), a Th2 cytokine (IL-4), inflammatory cytokines (IL-1α and TNF-α), and dendritic cell-inducible cytokines (IL-3 and GM-CSF). These cytokines in a proper combination augmented the anti-tumor effect of LH and prolonged survival time in Lewis lung carcinoma or B16 melanoma significantly. Moreover, the 12-cytokine cocktail suppressed B16 metastasis to the lung and lymph nodes, and complete regression of the tumors without regrowth occurred in 3 of 5 mice. In the cured three B16 mice, there was hyperplasia of lymphatic organs with many CD3-positive T lymphocytes. The most effective cytokine combination should be able to augment the anti-tumor effect of other therapies besides hyperthermia that induce the necrosis of tumor cells.     

Characterization of factors regulating successful immunotherapy using a tumor-specific cytotoxic T lymphocyte clone: Role of interleukin-2, cycling pattern of lytic activity and adhesion molecules

Adoptive immunotherapy against cancer has met with varying degrees of success, the reasons for which remain unclear. The present study characterizes factors that regulate successful immunotherapy of mice bearing a syngeneic T-cell lymphoma, designated LSA, using a tumor-specific cytotoxic T lymphocyte (CTL) clone, PE-9. Adoptive transfer of PE-9 cells afforded significant protection in normal but not in nude mice against LSA tumor. However, the PE-9 cells could protect the nude mice when injected along with normal CD4⁺ T cells. Administration of IL-2 along with PE-9 cells failed to enhance tumor immunotherapy. IL-2 therapy was toxic inasmuch as injection of the CTL clone PE-9 + IL-2, but not PE-9 or IL-2 alone, for 5 days into irradiated mice caused vascular leak syndrome (VLS). PE-9 cells cultured with high doses of rIL-2 in vitro also caused TCR-independent and MHC-unrestricted lysis of SV40-transformed endothelial cells. Furthermore, PE-9 cells cultured in vitro for 24-96 hr with IL-2 exhibited cycling pattern of tumorspecific cytotoxicity with maximum cytotoxicity demonstrable at 48 hr and virtually no cytolytic activity at 96 hr of culture or thereafter. The loss of cytotoxicity correlated with downregulation of several adhesion molecule expressions on PE-9 cells, particularly the αβ-TCR, as well as the mRNA for TNF-α, IFN-γ and perforin, although the levels of granzyme A were not altered. Interestingly, the outcome of immunotherapy by PE-9 cells depended on the cycling pattern of cytotoxicity. Our data suggest that successful immunotherapy against cancer using a CTL clone depends on several factors, such as the cycling pattern of lytic activity, density of adhesion molecules, levels of cytokines expressed and the ability of IL-2 and CTL to trigger VLS.     

Egfl7 promotes tumor escape from immunity by repressing endothelial cell activation

Downregulating the leukocyte adhesion molecules expressed by endothelial cells that line tumor blood vessels can limit the entry of immune effector cells into the tumor mass, thereby contributing to tumoral immune escape. Egfl7 (also known as VE-statin) is a secreted protein specifically expressed by endothelial cells in normal tissues and by cancer cells in various human tumors. High levels of Egfl7 correlate with higher tumor grade and poorer prognosis. Here we show that expression of Egfl7 in breast and lung carcinoma cells accelerates tumor growth and metastasis in immunocompetent mice but not in immunodeficient mice. Tumors expressing Egfl7 were infiltrated relatively poorly by immune cells and were characterized by reduced levels of immunostimulatory cytokines [IFN-γ, interleukin-12 (IL-12)] and fewer endothelial adhesion molecules [intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)]. In vitro studies revealed that Egfl7 inhibited the expression of leukocyte adhesion molecules by endothelial cells, preventing lymphocyte adhesion. In contrast, Egfl7 did not exert any effects on immune cell activation. Human breast cancer lesions expressing high levels of Egfl7 also expressed less ICAM-1 and VCAM-1 in their blood vessels, also indicating an inverse correlation between expression levels of Egfl7 and IFN-γ. Thus, Egfl7 expression in tumors promotes tumor progression by reducing the expression of endothelial molecules that mediate immune cell infiltration. Our findings highlight a novel mechanism through which tumors escape immune control.     

Hyperthermia decreases cytokine-mediated adhesion molecule expression on human umbilical vein endothelial cells

Although hyperthermia has been used as an effective cancer treatment modality, its effects on metastasis of tumour cells are not clear. Since adhesion molecules play a key role in metastasis, we evaluated how the expression of adhesion molecules is influenced by hyperthermia. Human umbilical vein endothelial cells were incubated in vitro for 1 h. at 39, 42, 43 and 44°C with and without addition of tumour-necrosis factor (TNF) or interferon-gamma (IFN-γ) and the expression of endothelial cell leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class-II molecule was measured. Expression of MHC class-II molecules and expression of unstimulated constituent ICAM-1's was not reduced by heat treatment. In contrast, expression of cytokine-induced ELAM-1's and ICAM-1's was significantly lower after heat treatment. The adhesion to HUVEC in vitro of HL-60 leukemia cells, which express sialyl-Lewis-x antigen as a ligand to ELAM-1, was diminished after incubation at 42°C and totally lost after treatment at 44°C. This suggests that any decrease in metastasis formation after heat treatment, which is occasionally observed, could be due to a reduced action of TNF or related cytokines on adhesion molecule induction and subsequent membrane expression by the endothelial cell. A possible underlying mechanism involved is a heat-induced alteration or blockage of the biosynthetic pathways required for synthesis of ELAM-1 and ICAM-1 proteins.     

CD3 directed bispecific antibodies induce increased lymphocyte-endothelial cell interactions in vitro

Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells. Intravenous administration of BIS-1 F(ab')2 to carcinoma patients in a phase I/II clinical trial, caused immunomodulation as demonstrated by a rapid lymphopenia prior to a rise in plasma tumour necrosis factor-alpha and interferon-gamma levels. Yet, no lymphocyte accumulation in the tumour tissue and no anti-tumour effect could be observed. These data suggest a BsMAb-induced lymphocyte adhesion to blood vessel walls and/or generalized redistribution of the lymphocytes into tissues. In this study, we describe the effects of BIS-1 F(ab')2 binding to peripheral blood mononuclear cells (PBMC) on their capacity to interact with resting endothelial cells in vitro. Resting and pre-activated PBMC exhibited a significant increase in adhesive interaction with endothelial cells when preincubated with BIS-1 F(ab')2, followed by an increase in transendothelial migration (tem). Binding of BIS-1 F(ab')2 to PBMC affected the expression of a number of adhesion molecules involved in lymphocyte adhesion/migration. Furthermore, PBMC preincubated with BIS-1 F(ab')2 induced the expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 during adhesion/tem. These phenomena were related to the CD3 recognizing antibody fragment of the BsMAb and dependent on lymphocyte-endothelial cell contact. Possibly, in patients, the BIS-1 F(ab')2 infusion induced lymphopenia is a result of generalized activation of endothelial cells, leading to the formation of a temporary sink for lymphocytes. This process may distract the lymphocytes from homing to the tumour cells, and hence prevent the occurrence of BIS-1 F(ab')2 - CTL-mediated tumour cell lysis.     

Comparison of response to radiation,hyperthermia and cisplatin in parental and polymerase β knockout cells

The role of polymerase &#103 in response to radiation, cisplatin and hyperthermia was examined in a pair of mouse cell lines, comprising a normal parental line and a derivative with polymerase &#103 knockout. Cell survival was assessed using the colony survival assay. For irradiation, there was no difference in response between the two cell lines. Treatment with cisplatin for 1h showed a large increase in resistance in the mutant cell line. The results with hyperthermia were more complex. The mutant was more resistant to 45°C heating, but was slightly more heat sensitive than the wild type at 41°C. Thus, in summary, while the knockout of polymerase &#103 did not alter radiation sensitivity, it did increase resistance to cisplatin and induced resistance to hyperthermia at higher temperatures (45°C).     

Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression,an effect amplified by interleukin-1β

The Notch1 and Notch4 signaling pathways regulate endothelial cell homeostasis. Inflammatory cytokines induce the expression of endothelial adhesion molecules, including VCAM1, partly by downregulating Notch4 signaling. We investigated the role of endothelial Notch1 in this IL-1β-mediated process. Brief treatment with IL-1β upregulated endothelial VCAM1 and Notch ligand Jagged1. IL-1β decreased Notch1 mRNA levels, but levels of the active Notch1ICD protein remained constant. IL-1β-mediated VCAM1 induction was downregulated in endothelial cells subjected to pretreatment with a pharmacological inhibitor of the γ-secretase, which activates Notch receptors, producing NotchICD. It was also downregulated in cells in which Notch1 and/or Jagged1 were silenced.Conversely, the forced expression of Notch1ICD in naïve endothelial cells upregulated VCAM1 per se and amplified IL-1β-mediated VCAM1 induction. Jagged1 levels increased and Notch4 signaling was downregulated in parallel. Finally, Notch1ICD and Jagged1 expression was upregulated in the endothelium of the liver in a model of chronic liver inflammation.In conclusion, we describe here a cell-autonomous, pro-inflammatory endothelial Notch1-Jagged1 circuit (i) triggering the expression of VCAM1 even in the absence of inflammatory cytokines and (ii) enhancing the effects of IL-1β. Thus, IL-1β regulates Notch1 and Notch4 activity in opposite directions, consistent with a selective targeting of Notch1 in inflamed endothelium.     

Water immersion pretreatment decreases pro-inflammatory cytokine production in cholecystokinin-octapeptide-induced acute pancreatitis in rats: possible role of HSP72

Heat shock proteins (HSPs) are cytoprotective proteins that are expressed constitutively and/or at elevated levels upon the exposure of cells to stress. The aim of this study was to investigate the potential effects of HSP preinduction by cold- (CWI) or hot-water immersion (HWI) on pro-inflammatory cytokine production (IL-1, IL-6, TNF- &#102 ) in cholecystokininoctapeptide(CCK)-induced acute pancreatitis. Rats were injected with 3 75µg/kg CCK subcutaneously at intervals of 2h at the peak level of HSP synthesis, as determined by Western blot analysis. The animals were killed by exsanguination through the abdominal aorta 2h after the last CCK injection. The serum IL-1, IL-6, TNF- &#102 , and amylase levels, the pancreatic weight/body weight ratio, and the pancreatic contents of DNA, protein, amylase, lipase and trypsinogen were measured; biopsy for histology was taken. HWI significantly elevated the HSP72 expression, while CWI significantly increased the HSP60 expression. HWI pretreatment decreased all of the measured serum cytokine levels in this acute pancreatitis model. CWI and HWI pretreatment ameliorated most of the examined laboratory and morphological parameters of CCK-induced pancreatitis. The findings suggest the possible roles of HSP60 and HSP72 in the protection against CCK-induced pancreatitis. HSP72 might also participate in the reduction of pro-inflammatory cytokine synthesis.     

Role of Interleukin-2 Activated MHC-Nonrestricted Lymphocytes in Antileukemia Activity and Therapy

Although the application of biological therapy for solid tumors with cytokines and adoptively transferred activated lymphocytes has received substantial attention, this approach has not been actively explored in treatment of hematopoietic neoplasms. This review will address the possibilities of interleukin-2 (IL-2) and IL-2 activated natural killer (NK) cells and T cells in antileukemia reactivity and therapy. The new approaches to optimal activation and generation of oncolytic cells, selective propagation of lymphocyte subsets, and the role of adhesion molecules in antileukemia cytotoxicity will also be addressed. We trust that this article will be conducive to the development of new directions in leukemia research and treatment.     

Role of interleukin-2 activated MHC-nonrestricted lymphocytes in antileukemia activity and therapy.

Although the application of biological therapy for solid tumors with cytokines and adoptively transferred activated lymphocytes has received substantial attention, this approach has not been actively explored in treatment of hematopoietic neoplasms. This review will address the possibilities of interleukin-2 (IL-2) and IL-2 activated natural killer (NK) cells and T cells in antileukemia reactivity and therapy. The new approaches to optimal activation and generation of oncolytic cells, selective propagation of lymphocyte subsets, and the role of adhesion molecules in antileukemia cytotoxicity will also be addressed. We trust that this article will be conducive to the development of new directions in leukemia research and treatment.     

The effects of hyperthermia on the immunomodulatory properties of human umbilical cord vein mesenchymal stem cells (MSCs)

Purpose: Hyperthermia can modulate inflammation and the immune response. Based on the recruitment of mesenchymal stem cells (MSCs) to inflamed tissues and the immunomodulatory properties of these cells, the aim of this study was to examine the effects of hyperthermia on the immunomodulatory properties of MSCs in a mixed lymphocyte reaction (MLR).

Materials and methods: Passages 4–6 of human umbilical cord vein mesenchymal stem cells were co-cultured in a two-way MLR. Cells in the hyperthermia groups were incubated at 41?°C for 45?min. A colorimetric assay was employed to examine the effects of MSCs on cell proliferation. The levels of IL-4 and TNF-α proteins in the cell culture supernatant were measured, and non-adherent cells were used for RNA extraction, which was then used for cDNA synthesis. RT-PCR was utilised to assess levels of IL-10, IL-17A, IL-4, TNF-α, TGF-β1, FOX P₃, IFN-γ, CXCL12 and β-actin mRNA expression.

Results: UCV-MSCs co-cultured in an MLR reduced lymphocyte proliferation at 37?°C, whereas hyperthermia attenuated this effect. Hyperthermia increased expression of IL-10, TGF-β1 and FOXP3 mRNAs in co-culture; however, no effects on IL-17A and IFN-γ were observed, and it reduced CXCL12 expression. In co-culture, IL-4 mRNA and protein increased at 37?°C, an effect that was reduced by hyperthermia. No considerable change in TNF-α mRNA expression was found in hyperthermia-treated cells.

Conclusion: Hyperthermia increases cell proliferation of the peripheral blood mononuclear cells and modifies the cytokine profile in the presence of UCV-MSCs.