（1.第二軍醫大學附屬長海醫院放療科，上海 200433；2.上海第二醫科大學附屬瑞金醫院腫瘤放化療科，上海 200025；3.解放軍第88醫院核醫學科，山東泰安 271000）
摘要：發熱溫度範圍全身熱療（FR-WBH）可提高免疫效應細胞活性、誘導免疫效應細胞再分布以及影響某些細胞因子和熱休克蛋白表達，具有重要的免疫調節作用，是一種安全、有效的輔助性腫瘤治療手段。
近年來，隨著熱療的生理學和病理生理學研究逐步深入，以及更為安全的加溫設備和更為精確的測溫、控溫技術的應用，全身熱療（Whole body hyperthermia, WBH）得到了迅速發展，已成為一種重要的輔助性腫瘤治療手段。
目前臨床常用的全身熱療方式主要有兩種：高溫短時間全身熱療（High-temperature short-duration WBH，HS-WBH），即將體中心溫度升高至41.8℃、持續1~2小時，和低溫長時間全身熱療（Low-temperature long-duration WBH, LL-WBH），體中心溫度升高至39.5~40℃、持續6小時或以上。後者類似於生理狀態下機體對感染的保護性反應，故又稱為發熱溫度範圍全身熱療（Fever-range WBH, FR-WBH）。研究表明，FR-WBH對機體免疫功能有重要的調節作用。
1 提高免疫效應細胞的活性
在小鼠離體和在體實驗中均發現，FR-WBH可激活蛋白激酶C（PKC）並刺激產生多種PKC同工酶，PKC特異性抑制劑Calphostin C可抑制此作用，提示FR-WBH可調節細胞信號傳導途徑中與淋巴細胞增殖和活化相關的關鍵步驟。而且加溫後淋巴細胞以血影蛋白（spectrin）為基礎的細胞骨架結構亦明顯改變，細胞極性增加，偽足形狀改變，免疫活性提高［1、2］。對人臍血和成人外周血T淋巴細胞進行體外加溫（≤40℃）後，其細胞毒活性亦提高［3］。
表皮Langerhans細胞是重要的抗原提呈細胞（APC），由其激活的T淋巴細胞介導的免疫反應在機體抗腫瘤免疫中起重要作用。研究發現，FR-WBH後，Langerhans細胞出現更多的斑點狀結構，樹突減少，而且向淋巴結引流增加，能更有效地提呈腫瘤抗原給效應細胞［4］。健康志願者接受FR-WBH後，其單核細胞表面內毒素受體CD14和補體受體CD11b的表達均升高，在脂多糖刺激下釋放的腫瘤壞死因子（TNF）-α亦增加，證明FR-WBH可直接激活單核細胞，使其對內毒素的反應性提高［5］。
2 誘導免疫效應細胞再分布
對不同品系、不同腫瘤類型荷瘤鼠的動物實驗表明，FR-WBH後，外周血、脾和腹腔中白細胞數量明顯降低，以淋巴細胞減少為主［6］。晚期實體瘤患者的FR-WBH臨床I期試驗亦見治療後循環中T淋巴細胞和L-選擇素(+)淋巴細胞數量減少［7］。而腫瘤血管直徑明顯擴大，腫瘤血管和腫瘤基質內有核細胞如類淋巴細胞、巨噬細胞、自然殺傷（NK）細胞及粒細胞等浸潤增加，表明FR-WBH可促使免疫效應細胞向腫瘤內轉移，其中NK細胞與腫瘤細胞的凋亡關係密切［8］。
同時，FR-WBH可促使淋巴細胞向二級淋巴組織（如外周淋巴結、腸繫膜淋巴結和Peyer’s結等）歸巢增加，這種增加是通過提高循環中淋巴細胞與腫瘤組織和淋巴組織中特殊的高內皮細胞小靜脈（HEV）之間的依賴L-選擇素（L-selectin）及α4β7整合素（integrin）的黏附作用實現的，在正常組織血管的鱗狀內皮細胞中則無此現象［9、10］。
進一步的研究顯示，FR-WBH促進淋巴細胞與HEV黏附是通過提高L-選擇素和α4β7整合素的活性實現的，並未改變其細胞表面密度，而且這種黏附作用的提高不僅在體內，而且在體外實驗中亦能觀察到，表明加溫引起的此種效應在局部淋巴組織微環境中即可出現，不需要下丘腦-垂體-腎上腺軸的參與［11、12、13］。另一方面，FR-WBH可增強內皮細胞肌動蛋白的聚合，提高內皮細胞分泌的能刺激α4β7整合素淋巴細胞歸巢受體的因子的活性，從而使大量淋巴細胞選擇性地轉移至感染或腫瘤部位，放大免疫效應［14］。
3 對細胞因子表達的影響
實驗材料不同、條件不同，FR-WBH對細胞因子表達的影響亦不同。離體時FR-WBH對大血管及小血管內皮細胞黏附分子（ICAM-1, E-selectin, VCAM-1, P-selectin, PECAM-1, PNAd, MAdCAM-1）的表達、細胞因子（IL-1β, TNF-α, IFN-γ, IL-6, IL-11, IL-12, IL-13）的釋放和趨化因子（IL-8, RANTES, MCP-1, MIP-1β, MIG）的分泌均無影響［14］。小鼠腹膜內注射脂多糖後熱療，血清中TNF-α、IL-6、急性期蛋白（APPs）α1-酸性糖蛋白和結合珠蛋白（haptoglobin）濃度升高，而單用FR-WBH則無此影響［15］。成人臍靜脈內皮細胞FR-WBH後PECAM-1分泌增加，24小時後E-選擇素和RANTES亦有增加，VEGF分泌則減少［16］。
4 對熱休克蛋白的影響
熱應激可誘導熱休克蛋白（HSPs）和糖調蛋白（GRPs）表達上調，保護細胞自身免受損傷。實驗發現，FR-WBH可提高小鼠心、腎、肺、淋巴結和胸腺組織中HSP70及肺、淋巴結和胸腺組織中HSP110的表達，其中在淋巴組織中表達上調最高，可能與加溫後免疫應答提高有關。糖調蛋白（GRP170）表達則不受影響［17、18］。
人外周血離體加溫後HSP70表達的變化與溫度和細胞類型有關，加溫至39℃時即在單核細胞中有明顯誘導，大於41℃時僅在淋巴細胞和多形核白細胞中有輕微升高。但健康志願者FR-WBH後HSP70的表達在所有類型淋巴細胞中均有同等程度的升高，並無離體實驗時表現出的不同細胞類型間的差異［19］。
5 與免疫治療的協同作用
對人成膠質細胞的體外實驗表明，加溫至39℃可明顯提高干擾素（IFN）的抗病毒活性及對不同腫瘤細胞的抗增殖作用，FR-WBH與IFN聯合應用對於免疫力低下的晚期惡性腫瘤患者有治療價值［20］。FR-WBH可抑制VEGF產生，與TNF聯合應用可增強其抗血管生成作用［16］。另有研究發現，FR-WBH可明顯提高HSP110和HSP70作為腫瘤特異性疫苗的抗腫瘤活性［18］。FR-WBH亦能提高荷實體瘤小鼠的放射免疫治療效果。皮下移植了人結腸癌的裸鼠FR-WBH後靜脈注射放射性碘標記的抗CEA單克隆抗體，雖未改變抗體的生物學分布，但可提高腫瘤的放射敏感性，明顯抑制腫瘤的生長［21］。
6 結論
作為一種新的腫瘤治療方法，FR-WBH可顯著增強機體免疫功能，提高放療、化療和免疫治療的效果，且安全無毒，尤其適用於免疫力低下的晚期惡性腫瘤患者。其最優的治療方案、與其他腫瘤治療手段的最佳結合方式尚需進一步研究。
Influence of fever-range whole body hyperthermia on immune function
CHEN Hong-lei1 JIN Ye-ning2 PENG Yong3
(1. Department of Radiation Oncology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China; 2.Deparment of Oncology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China; 3.Department of Nuclear Medicine, the 88th Hospital of PLA, Tai』an City, Shangdong 271000, China.)
Abstract: Fever-range whole body hyperthermia (FR-WBH) has an important immunoregulatory effect: enhance the activity of immune effector cells, induce immune effector cells redistribution, and affect the expression of some cytokines and heat shock proteins. This treatment protocol could be used as an effective and nontoxic adjuvant to other anti-tumor therapies.
With advances in physiology and pathophysiology of hyperthermia and applications of safer devices and more accurate temperature measure and control techniques, whole body hyperthermia (WBH) developed rapidly in recent years. It has became an important adjuvant anti-tumor treatment.
Two predominant WBH protocols presently being used in the clinic include a high-temperature, short-duration (HS) WBH, where core body temperature is raised to 41.8℃ for 1~2h, and a low-temperature, long-duration (LL) WBH, where core body temperature is raised to 39.5~40℃ for 6h or more. The latter protocol, comparable to the protective physiological reaction during infection, is also called fever-range (FR) WBH. Studies have revealed that FR-WBH has an important immunoregulatory effect on the host.
1 Enhance the activities of immune effector cells
FR-WBH treatment of mice, in vitro and in vivo, resulted in a activation of protein kinase C (PKC) and induced many PKC isozymes. Calphostin C, a specific inhibitor of PKC, could inhibit this action. This suggested that FR-WBH can modulate critical steps in the signal transduction pathways necessary for lymphocytes activation and proliferation. Furthermore, the spectrin-based cytoskeleton of lymphocytes also altered obviously after hyperthermia, which caused the cell polarity increased, the uropods formation changed, and the immune activity of lymphocytes enhanced［1、2］. Cytotoxic activities of T-lymphocytes from adult blood as well as from cord blood could be enhanced at febrile range (≤40℃)［3］.
The Langerhans cells (LCs) of epidermis are important antigen-presenting cells (APC). The T-lymphocytes-mediated immune response initialed by LCs plays an important role in host anti-tumor immune. Studies had showed that LCs exhibited a more punctate morphology with fewer dendritic processes after FR-WBH treatment. Furthermore, the numbers of LCs drained into the lymph nodes increased, presenting the tumor antigens to effector cells more effectively［4］.The expression of the monocyte endotoxin receptor CD14 and the complement receptor CD11b increased in healthy volunteers after FR-WBH. The TNF-α release stimulated by lipopolysaccharide also increased. This supported that FR-WBH can directly activate monocytes, which enhance their ability to respond to endotoxin［5］.
2 Induce redistribution of immune effector cells
Various studies in animal tumor models revealed that leukocyte numbers in the peripheral blood, spleen and peritoneal cavity are significantly decreased immediately after FR-WBH treatment, reflecting the decrease in lymphocyte numbers［6］.A phase I clinical trial of patients with advanced solid tumors showed that the numbers of circulating T-lymphocytes and L-selectin position lymphocytes were decreased after FR-WBH treatment［7］. However, the diameter of blood vessels within the tumor were obviously expended. The increased numbers of nucleated blood cells , such as lymphocyto-like cells, macrophages, natural killer (NK) cells and granulocytes, were observed in the tumor vasculature and in the tumor stroma. This indicates that immunoeffector cells may have greater access to the interior of the tumor under the FR-WBH stimulation. And the tumor cell apoptosis appears to be due largely to the NK cells［8］.
Meanwhile, FR-WBH stimulated lymphocytes homing to secondary lymphoid tissues, such as peripheral lymph nodes, mesenteric lymph nodes and Peyer’s patch, by increasing L-selectin and α4β7 integrin-dependent adhesive interactions between circulating lymphocytes and specialized high endothelial venules (HEV) in lymphoid tissues and tumor tissues. In contrast, FR-WBH did not augment adhesion in squamous endothelium of normal blood vessels［9、10］.
Further studies revealed that FR-WBH stimulates adhesion between HEV and lymphocytes by enhancing the activities of L-selectin and α4β7 integrin rather than their cell surface density. And similar increases also occurred in vitro, indicating that the local lymphoid tissue microenvironment is sufficient for this thermic response, not requiring involvement of hypothalamus-pituitary-adrenal axis［11、12、13］.On the other hand, FR-WBH augmented actin polymerization in endithelial cells and enhanced the ability of endothelial-derived factors to transactivate theα4β7 integrin lymphocyte homing receptor. Massive lymphocytes selectively migrate to the sites of infection or tumor, amplifying the immune response［14］.
3 Effects on cytokines expression
Under different environments, the effects of FR-WBH on cytokines expression are also different. In vitro, fever-range hyperthermia did not affect the expression of adhesion molecules (ICAM-1, E-selectin, VCAM-1, P-selectin, PECAM-1, PNAd, MAdCAM-1), the release of cytokines (IL-1β, TNF-α, IFN-γ, IL-6, IL-11, IL-12, IL-13) and the secretion of chemokines (IL-8, RANTES, MCP-1, MIP-1β, MIG)［14］.When mice received FR-WBH just after intraperitoneal administration of lipopolysaccharide, serum concentrations of TNF-α, IL-6, the acute phase proteins (APPs)α1-acid glycoprotein and heptoglobin increased. FR-WBH alone did not affect serum concentrations of these cytokines［15］.FR-WBH treatment increased the PECAM-1 production in human umbilical vein endothelial cells. Increased E-selectin and RANTES levels were also observed at 24h after treatment. However, the production of VEGF decreased［16］.
4 Effects on heat shock proteins
Heat stress can induce the upregulation of heat shock proteins (HSPs) or glucose regulated proteins (GRPs) expression, which help protect cells against damages. Studies showed that, FR-WBH treatment of mice did enhance HSP expression: HSP70 in heart, kidney, lung, lymph nodes and thymus; and HSP110 in lung lymph nodes and thymus. The lymphoid tissues displayed the most consistent upregulation of both HSP70 and HSP110. This may relate to the enhanced immune response during febrile temperatures. In contrast, expression of GRP170 was not affected by FR-WBH in any tissues［17、18］.
Heat treatment of human peripheral blood in vitro showed that the change of HSP70 expression depends on temperature and cell type. At 39℃, a strong HSP70 induction was seen in monocytes. Up to 41℃, HSP70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in healthy volunteers, HSP70 expression enhanced in all kinds of lymphocytes with comparable after FR-WBH, without the cell type-specific variations as observed in vitro［19］.
5 Cooperation with immunotherapy
Treatment of human fibroblast cells with fever-range hyperthermia (39℃) in vitro significantly enhanced the antiviral activity and antiproliferative activity on different tumor cells of interferon (IFN). The combination of FR-WBH and IFN may have therapeutical values to late stage cancer patients who have a weaker immune system［20］.Fever-range hyperthermia decreased the production of VEGF. When combinated with TNF, the thrombotic effects on tumor neovasculature may be enhanced［16］.Another study showed that fever-range hyperthermia significantly enhanced the specific vaccine efficiency of HSP110 and HSP70［18］.FR-WBH also enhanced the therapeutic outcome of radio-immunotherapy for mice bearing solid cancers. After FR-WBH, nude mice bearing human colon cancer xenografts received intravenous injection of I-labelled anti-carcinoembryonic antigen (CEA) monoclonal antibody. Although hyperthermia did not alter the biodistribution of the antibody, the growth of tumor was significantly inhibited by increasing the radiosensitivity of the tumor［21］.
6 Conclusion
As a new anti-tumor treatment, FR-WBH can significantly enhance the host immune function and increase the therapeutic efficacy of radiotherapy, chemotherapy and immunotherapy without toxicity, especially applying to late stage cancer patients who have a weaker immune system. Its optimal treatment protocol and the best combined modality with other anti-tumor treatments are required further research.
References:
［1］ Di YP, Repasky EA, Subjeck JR. Distribution of HSP70, protein kinase C, and spectrin is altered in lymphocytes during a fever-like hyperthermia exposure[J]. J Cell Physiol, 1997, 172(1):44-54.
［2］ Wang XY, Ostberg JR, Repasky EA. Effect of fever-like whole-body hyperthermia on lymphocyte spectrin distribution, protein kinase C activity, and uropod formation[J]. J Immunol, 1999, 162(6):3378-3387.
［3］ Shen RN, Lu L, Young P, et al. Influence of elevated temperature on natural killer cell activity, lymphokine-activated killer cell activity and lectin-dependent cytotoxicity of human umbilical cord blood and adult blood cells[J]. Int J Radiat Oncol Biol Phys, 1994, 29(4):821-826.
［4］ Ostberg JR, Patel R, Repasky EA. Regulation of immune activity by mild (fever-range) whole body hyperthermia: effects on epidermal Langerhans cells[J]. Cell Stress Chaperones, 2000, 5(5):458-461.
［5］ Zellner M, Hergovics N, Roth E, et al. Human monocyte stimulation by experimental whole body hyperthermia[J]. Wien Klin Wochenschr, 2002, 114(3):102-107.
［6］ Ostberg JR, Repasky EA. Comparision of the effects of two different whole body hyperthermia protocols on the distribution of murine leukocyte populations[J]. Int J Hyperthermia, 2000, 16(1):29-43.
［7］ Kraybill WG, Olenki T, Evans SS, et al. A phaseⅠstudy of fever-range whole body hyperthermia (FR-WBH) in patients with advanced solid tumors: correlation with mouse models[J]. Int J Hyperthermia, 2002, 18(3):253-266.
［8］ Burd R, Dziedzic TS, Xu y, et al. Tumor cell apoptosis, lymphocyte recruitment and tumor vascular changes are induced by low temperature, long duration (fever-like) whole body hyperthermia[J]. J Cell Physiol, 1998, 177(1):137-147.
［9］ Evans SS, Wang WC, Bain MD, et al. Fever-range hyperthermia dynamically regulates lymphocyte delivery to high endothelial venules[J]. Blood, 2001, 97(9):2727-2733.
［10］ Ostberg JR, Gellin C, Patel R, et al. Regulatory potential of fever-range whole body hyperthermia on Langerhans cells and lymphocytes in an antigen-dependent cellular immune response[J]. J Immunol, 2001, 167(5):2666-2670.
［11］ Wang WC, Goldman LM, Schleider DM, et al. Fever-range hyperthermia enhance L-selectin-dependent adhesion of lymphocytes to vascular endothelium[J]. J Immunol, 1998, 160(2):961-969.
［12］ Evans SS, Bain MD, Wang WC. Fever-range hyperthermia stimulates alpha4beta7 integrin-dependent lymphocyte-endothelial adhesion[J]. Int J Hyperthermia, 2000, 16(1):45-59.
［13］ Evanss SS, Schleider DM, Bowman LA, et al. Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix[J]. J Immunol, 1999, 162(6):3615-3624.
［14］ Shah A, Unger E, Bain MD, et al. Cytokine and adhesion molecule expression in primary human endothelial cells stimulated with fever-range hyperthermia[J]. Int J Hyperthermia, 2002, 18(6):534-551.
［15］ Ostberg JR, Taylor SL, Baumann H, et al. Regulatory effects of fever-range whole-body hyperthermia on the LPS-induced acute inflammatory response[J]. J Leukoc Biol, 2000, 68(6):815-820.
［16］ Gnantf MF, Turner EM, Alexander HR. Effects of hyperthermia and tumour necrosis factor on inflammatory cytokine secretion and procoagulant activity in endothelial cells[J]. Cytokine, 2000, 4(4):339-347.
［17］ Ostberg JR, Kaplan KC, Repasky EA. Induction of stress proteins in a panel of mouse tissues by fever-range whole body hyperthermia[J]. Int J Hyperthermia, 2002, 18(6):552-562.
［18］ Wang XY, Kazim L, Repasky EA, et al. Characterization of heat shock protein 110 and glucose-regulated protein 170 as cancer vaccines and the effects of fever-range hyperthermia on vaccine activity[J]. J Immunol, 2001, 166(1):490-497.
［19］ Oehler R, Pusch E, Zellner M, et al. Cell type-specific varivations in the induction of hsp70 in human leukocytes by feverlike whole body hyperthermia[J]. Cell Stress Chaperones, 2001, 6(4):306-315.
［20］ Payne J, Nair MP, Ambrus JL, et al. Mild hyperthermia modulates biological activities of interferons[J]. Int J Hyperthermia, 2000, 16(6):492-507.
［21］ Saga T, Sakahara H, Nakamoto Y, et al. Enhancement of the therapeutic outcome of radio-immunotherapy by combination with whole-body mild hyperthermia[J]. Eur J Cancer, 2001, 37(11):1429-1434.
(中華熱療學會 )