Antibodies to 70 kD and 90 kD heat shock proteins are associated with graft-versus-host disease in peripheral blood stem cell transplant recipients

The development of graft-versus-host disease (GVHD) can be modified by non-MHC factors. Based on our previous studies that showed an involvement of 70kD heat shock protein (hsp70) in the pathology of acute GVHD in a rat model, we determined serum levels of antibodies to hsp70, hsp90 and hsp60 in human recipients after allogeneic peripheral blood stem cell transplantation (PBSCT). Serum levels of these antibodies were correlated with GVHD status in the recipients. Twenty-nine recipients with high-risk haematological malignances, who received G-CSF mobilized allogeneic PBSCT from HLA matched family donors, were evaluated between 30 and 960 days after transplantation. Two recipients had no GVHD, 18 developed acute followed by chronic GVHD and nine developed only chronic GVHD. Patients with acute GVHD had a significant increase in IgM anti-hsp70 and/or anti-hsp90 early (30-90 days) after transplantation. In addition, an increase in IgM anti-hsp70 and/or anti-hsp90 antibodies preceded or accompanied chronic GVHD. Antibody levels returned to normal within the next 400 days in the majority of patients. Anti-hsp60 antibody levels were not different from control levels regardless of GVHD status. This study implies that the development of acute and/or chronic GVHD in humans is accompanied by an increase in anti-hsp70 and anti-hsp90 antibodies. Monitoring levels of anti-hsp70 and anti-hsp90 antibodies in stem cell transplant recipients may serve as a diagnostic tool and help to predict the onset of GVHD.     

Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway

Dendritic cells (DC) are key components of innate and adaptive immune responses. The identity of endogenous signals that activate DC is a crucial and unresolved question. We report here that heat shock proteins (HSP), the most abundant and conserved mammalian molecules, constitute such an internal signal. Necrotic but not apoptotic cell death leads to release of HSP gp96, calreticulin, hsp90 and hsp70. HSP stimulate macrophages to secrete cytokines, and induce expression of antigen-presenting and co-stimulatory molecules on the DC. The HSP gp96 and hsp70 act differentially, and each induces some but not all molecules. HSP interact with these antigen-presenting cells through the highly conserved NF-kappa B pathway. As HSP are intracellular, abundant and soluble, their presence in the extra-cellular milieu and the consequent activation of antigen-presenting cells (APC) constitutes an excellent mechanism for response to cell death. As HSP are conserved from bacteria to mammals, the ability of HSP to activate APC provides a unified mechanism for response to internal and external stimuli.     

A 71-kD heat shock protein (hsp) from Mycobacterium tuberculosis has modulatory effects on experimental rat arthritis

The effects of a mycobacterial 71-kD hsp antigen have been investigated for its ability to modulate arthritis in rats. Subcutaneous injection (base of tail) of increasing amounts of hsp71 from Mycobacterium tuberculosis (MTB) produced dose-dependent differential inhibitory effects on induction of arthritis by MTB and CP20961 in rats. As little as 1 μg of the hsp71 produced a reduction in MTB arthritis, whereas complete protection was observed when 50 μg were administered. When 71-kD-treated rats were challenged with CP20961, all developed reduced symptoms of arthritis compared with control rats, but in this model no complete protection was observed over the dose range studied. The effects of 71-kD pretreatment on collagen II arthritis were not significant, but in general symptoms of arthritis were milder than in the control group. The same pattern of results was observed previously when hsp65 was used in the different models. These results show that the modulatory effects of hsp on adjuvant arthritis are not restricted to the hsp65 series, but are also mediated by a member of the hsp70 family.     

Mortalin/GRP75 promotes release of membrane vesicles from immune attacked cells and protection from complement-mediated lysis

The membrane attack complex (MAC) of the complement system is causing membrane damage and cell death. For protection, cells have adopted several resistance mechanisms, including removal of the membrane-inserted MAC by vesiculation. To identify proteins involved in MAC vesiculation, extracellular proteins released from K562 cells in response to treatment with sub-lytic complement were separated by acrylamide gel electrophoresis and protein bands were extracted, digested into peptides and the peptides were analyzed by mass spectrometry. A 75-kDa protein that was abundant in the supernatant of complement-treated cells was identified as mortalin/GRP75. Analysis by western blotting demonstrated that as early as 5 min after exposure to sub-lytic doses of complement, mortalin was released from K562 cells. Mortalin was released after complete activation of the complement system and formation of C5b-8, and even more so when C5b-9 was formed. Other pore formers, such as streptolysin O and melittin, did not induce release of mortalin. As shown, mortalin can bind to complement C8 and C9 and is shed in vesicles containing C9 and complement MACs. Anti-mortalin antibodies reduced mortalin release from complement-treated cells and elevated the extent of cell death by complement. Inhibitors of protein kinase C and extracellular signal-regulated protein kinase also prevented mortalin release from complement-activated cells. These results suggest that mortalin/GRP75 promotes the shedding of membrane vesicles loaded with complement MAC and protects cells from complement-mediated lysis.     

Protection of human breast cancer cells from complement-mediated lysis by expression of heterologous CD59

CD59, decay accelerating factor (DAF) and membrane cofactor protein (MCP) are widely expressed cell surface glycoproteins that protect host cells from the effects of homologous complement attack. Complement inhibitory activity of these proteins is species-selective. We show that the human breast cancer cell line MCF7 is relatively resistant to lysis by human complement, but is effectively lysed by rat or mouse complement. CD59, DAF and MCP were all shown to be expressed by MCF7. The species-selective nature of CD59 activity was used to demonstrate directly the effectiveness of CD59 at protecting cancer cells from complement-mediated lysis. cDNAs encoding rat and mouse CD59 were separately transfected into MCF7 cells, and cell populations expressing high levels of the rodent CD59 were isolated by cell sorting. Data show that rat and mouse CD59 were highly effective at protecting transfected MCF7 cells from lysis by rat and mouse complement, respectively. Data further reveal that rat CD59 is not effective against mouse complement, whereas mouse CD59 is effective against both mouse and rat complement. These studies establish a model system for relevant in vivo studies aimed at determining the effect of complement regulation on tumourigenesis, and show that for effective immunotherapy using complement-activating anti-tumour antibodies, the neutralization of CD59 and/or other complement inhibitory molecules will probably be required.     

Heat shock-enhanced T cell apoptosis with heat shock protein 70 on T cell surface in multicentric Castleman's disease

We report here that T cells from patients with multicentric Castleman's disease (MCD) are sensitive to hyperthermia. T cells from two of three patients with MCD revealed DNA ladder formation and chromatin condensation following heat shock (30 min at 41.5°C). Peripheral blood mononuclear cells (PBMC) from the same MCD patients exhibited high levels of spontaneous apoptosis after 72 h in culture and elevated apoptosis after heat shock, as evaluated by a quantitative flow cytometric assay. Heat shock protein 70 (hsp70) was detected on the cell surface of T cells in all three patients after heat shock. Furthermore, hsp70 was detected on T cells in the two MCD patients with apoptosis even in the absence of heat shock. T cells from normal samples did not show either heat-shock-induced expression of cell-surface hsp70 or apoptosis. Thus, heat shock treatment augmented hsp70 expression on the cell surface of T cells and enhanced apoptosis. Our studies suggest that hyperthermia may influence the clinical course of MCD.     

结核杆菌热休克蛋白肽段对胶原诱导性关节炎保护作用的研究

目的：研究结核杆菌HSP65肽段（P256-270）和结核杆菌HSP70肽段（P111-125）对类风湿关节炎动物模型——胶原诱导性关节炎（CIA）大鼠的保护性作用。方法：结核杆菌HSP肽段滴鼻免疫大鼠，观察其对CIA的防治效果，观察指标包括关节炎指数评分、关节病理学分析、酶联免疫斑点实验（ELISPOT）检测脾脏淋巴细胞分泌细胞因子（IL4、IFN-γ）水平、流式细胞术分析外周血T淋巴细胞亚群及酶联免疫吸附实验（ELISA）测定血清中抗CⅡ抗体水平。结果：分别经结核杆菌HSP65和HSP70肽段滴鼻免疫的实验组大鼠，与阳性对照组比较，关节炎指数明显下降（P〈0．05），病理变化减轻，抑制性细胞因子IL-4水平升高（分别为P〈0．05和P〈O．01），而炎性细胞因子IFN-1水平降低（P〈0．01），CD4^＋／CD8^＋T细胞比值降低（P〈0．05），血清中抗CIⅡ抗体水平也降低（分别为P〈0．05和P〈0．01）。结论：应用结核杆菌HSP65肽段或HSP70肽段能减轻CIA大鼠的症状和体征，其作用机制与调节T细胞的功能有关，此研究可为探讨人类RA新的免疫治疗方法提供依据。     

Induction of heat shock protein 70 reduces the alteration of striatal electrical activity caused by mitochondrial impairment

Since mild thermal stress seems to exert neuroprotection via induction of heat-shock protein 70 kDa (hsp70), we tested whether hsp70 would preserve striatal bioelectrical activity under conditions of mitochondrial impairment. Corticostriatal slices from rats that had undergone mild thermal stress were exposed to either rotenone or 3-nitropropionic acid (3-NP), that selectively inhibits mitochondrial complex I and complex II, respectively. Rotenone is utilized to obtain an experimental model of Parkinson's disease while 3-NP replicates Huntington's disease phenotype in experimental animals. The cerebral hsp70 increase did not alter field potential amplitude of the slices but partially protected them against rotenone-induced neurotoxicity. Similarly, induction of hsp70 had also a partial neuroprotective effect on the neurotoxicity caused by 3-NP on striatal field potential. Since rotenone and 3-NP treatments mimic the mitochondrial impairment and oxidative stress that contribute to development of Parkinson's disease and Huntington's disease, these data suggest that induction of hsp70 might represent a possible neuroprotective mechanism against the pathophysiological chain of events implicated in these neurodegenerative disorders.     

Enhanced sensitivity of P-glycoprotein-positive multidrug resistant tumor cells to complement-mediated lysis

The interaction of KB-V1, a multidrug resistant (MDR) variant of the KB-3–1 human oral carcinoma, with human complement was investigated. KB-V1 cells were found to be more sensitive than KB-3–1 cells to complement-mediated lysis. Detailed analysis of the capacity of KB cells to activate human complement demonstrated that both C3b deposition and formation of the membrane attack complex (MAC) are higher on KB-V1 than on KB-3–1 cells. Furthermore, the MAC formed on KB-V1 cells, but not on KB-3–1 cells, was found to be resistant to trypsin treatment, i.e. more stably inserted into the plasma membrane. Immunofluorescence analysis by flow cytometry showed that KB-V1 cells express less decay-accelerating factor (DAF, CD55) than KB-3–1 cells. Two other complement regulatory proteins, membrane cofactor protein (MCP, CD46) and CD59 are expressed to a similar extent on both KB-V1 and KB-3–1 cells. Treatment of KB-V1 cells with neutralizing anti-P-glycoprotein (P-gp) monoclonal antibodies reduced their sensitivity to complement. In addition, KB-V1 revertants which cease to express P-gp become more resistant to complement. These results indicate that multiple factors, such as reduced expression of DAF, enhanced deposition of C3b and increased binding and stability of the MAC may contribute to the increased complement sensitivity of KB-V1 cells. It is suggested that P-gp is responsible for the complement-sensitive phenotype of KB-V1 cells.     

High positive frequency of antibodies to metallothionein and heat shock protein 70 in sera of patients with metal allergy

Two principal types of stress protein, heat shock proteins (hsps) and metallothionein (MT), are induced in cells responding to a variety of stresses. They play an important role in protecting cells from these stresses. However, many reports indicate that antibodies to hsps are present in human serum and are associated with several autoimmunity diseases. Metals, which are commonly allergenic to humans, induce both MT and hsp70 (one of the hsps family). Until now, there has been no report of any antibody to MT in human serum. In the present study, serum samples from healthy controls (Group I), and patients suffering from atopic dermatitis without (Group II) or with (Group III) metal allergy, were measured for antibodies to MT and hsp70, using an enzyme-linked immunosorbent assay (ELISA). Metal allergy was confirmed by patch testing. We first found that antibody to MT exists in human serum. We also found a high positive frequency of antibody to MT (51.3%) and to hsp70 (43.6%) in the sera of Group III, compared to those of Group I (3.8% and 5.1%) or Group II (6.4% and 5.1%). Furthermore, there was a strong positive correlation between antibody to MT and antibody to hsp70 in Group III (P = 0.0013), but not in Group I and Group II. Our results indicate that antibody to MT exists in human serum, as do antibodies to hsps, and suggest that elevated levels of MT and hsp70 antibodies are associated with metal allergy in atopic patients.     

Heat shock protein 70 is involved in polaprezinc driven cell protection against Helicobacter pylori-induced injury

Polaprezinc (PZ) plays a role in the protection of gastric mucosa and inhibiting Helicobacter pylori (H. pylori) growth in vitro. The objective of this study was to determine the protective effects of PZ on human gastric epithelial cells (GES-1) against H. pylori-induced damage, while also examining heat shock protein 70 (HSP70) as a potential underlying factor in this protection. Our findings revealed that PZ exerted bactericidal effects against H. pylori strains. We also observed that PZ mitigated the H. pylori-induced damage to GES-1 cells by increasing cell viability, reducing LDH release, and decreasing the secretion of pro-inflammatory factors such as MCP-1 and IL-6. Co-culturing PZ with GES-1 cells significantly up-regulated the GES-1 HSP70 expression in both a time and dose-dependent manner. Pre-incubating (for 12 h) or co-culturing (for 24 h) GES-1 cells with PZ reversed the down-regulation of HSP70 in GES-1 cells caused by H. pylori infection. However, when quercetin was used to inhibit the up-regulation of HSP70 in GES-1 cells, the protective effect of PZ on GES-1 cells was significantly reduced. Based on the results of this study, PZ exhibits a protective role on GES-1 cells against H. pylori injury, as well as a direct bactericidal effect on H. pylori. HSP70 is involved in the PZ-driven host cell protection against H. pylori injury. These findings provide insight into alternative strategies for H. pylori treatment.     

Response to hepatocarcinoma Hca-F of mice immunized with heat shock protein 70 from elemene combo tumor cell vaccine

To analyze immune response to murine hepatocarcinoma Hca-F of mice immunized with heat shock protein 70 （HSP70） derived from elemene combo tumor cell vaccine （EC-TCV） of Hca-F, HSP70 was isolated from EC-TCV by ADP affinity chromatography. Mice were immunized with HSP70 intraperitoneally three times and spleen calls were sampled. For cells, their proliferation and cytotoxicity against Hca-F were measured with MTT assay and their phenotypes were analyzed with flow cytometry. Spleen cells of immunized mice with HSP70 exhibited more potent cytotoxicity against Hca-F and proliferation than that of normal control mice, but less potent than that of mice immunized with EC-TCV. Among three groups, the percent of T6 T lymphocytes in the mice immunized with HSP70 （35.5%） was the highest compared with 6.25% in normal mice, and 28.4% in the mice immunized with EC-TCV. Immunization of HSP70 derived from EC-TCV could elicit potent immune response to Hca-F. HSP70 is one of elements inducing anti-tumor immune responses against Hca-F.     

Involvement of the ERK mitogen-activated protein kinase in cell resistance to complement-mediated lysis

Sublytic doses of complement desensitize cells and make them resistant to lytic complement doses. This process, named complement-induced protection, requires calcium ion influx, protein kinase C activation and protein synthesis. The involvement of the extracellular signal-regulated kinase, ERK, in cell desensitization by sublytic complement was examined in erythroleukaemia K562 cells and in COS-7 cells. As shown here, ERK is activated in K562 and COS-7 cells within 10 min of sublytic immune attack and then shows a decline and a second peak of activation at 20 min. C7- and C8-deficient human sera have a small effect on ERK activity. However, a significant increase in ERK activation is observed when C7 or C8, respectively, is added back to these sera. Complement-induced ERK activation was blocked in cells treated with GF109203X or Go6976, two selective PKC inhibitors, as well as by treatment with PD098059, an inhibitor of MEK1, the ERK kinase. PD098059 treatment also sensitized K562 cells to complement-mediated lysis and prevented complement-induced protection. COS-7 cells transfected with a dominant-negative MEK plasmid were incapable of undergoing the process of complement-induced protection. In conclusion, cell desensitization by sublytic doses of the complement membrane attack complex involves a signalling cascade that includes PKC-mediated ERK activation.     

Identification and characterization of a DR4-restricted T cell epitope within chlamydia heat shock protein 60

An epitope within the 60 kD Chlamydia trachomatis heat shock protein (hsp) 60, recognized by a HLA-DRB1*0401-restricted T cell clone from a reactive arthritis patient, has been characterized. Stimulatory peptides contained a nine amino acid sequence (residues 38–46) predicted by algorithm to confer strong binding to DRB1*0401, with valine in the P1 position. The overall length of the peptide was critical for efficient recognition; peptides with at least one residue N-terminal to the putative P1 position were markedly more stimulatory than a peptide whose N-terminal is the P1 valine. Optimal responses were seen with 14mer peptides having two to three amino acids N- and C-terminal to the core 9mer. The sequence of the defined epitope is identical in hsp60 from both C. trachomatis and C. pneumoniae. Since the latter is a common respiratory pathogen, patients infected with C. trachomatis may already be primed for responses to hsp60 by prior infection with C. pneumoniae. Such secondary responses are important in the pathogenesis of chlamydia-induced inflammatory diseases such as trachoma. Priming by infection with enteric organisms was considered because of the similarity of the epitope sequence in Escherichia coli hsp60. However, although an E. coli-related peptide was recognized, intact E. coli hsp60 was not, suggesting that the epitope is cryptic in E. coli hsp60. Human hsp60 has six amino acid differences from chlamydial hsp60 in the epitope sequence and was not recognized. Thus cross-reactive recognition of self hsp60 could not be implicated in the pathogenesis of chlamydia-induced reactive arthritis in this patient.     

Induction of heat shock proteins fails to produce protection against trypsin-induced acute pancreatitis in rats

Heat shock proteins (HSPs) are necessary in the synthesis, degradation, folding, transport, and translocation of different proteins. It is well known that the increased expression of HSPs may have a protective effect against cerulein-induced pancreatitis in rats or against choline-deficient ethionine-supplemented diet model pancreatitis in mice. The aim of this study was to investigate the potential effects of HSP preinduction by cold or hot water immersion on trypsin-induced acute pancreatitis in rats. Trypsin was injected into the interlobular tissue of the duodenal part of the pancreas at the peak level of HSP synthesis, as determined by Western blot analysis. The rats were sacrificed by exsanguination through the abdominal aorta 6 h after the trypsin injection. The serum amylase activity, the tumor necrosis factor-α, interleukin-1, and interleukin-6 levels, the pancreatic weight/body weight ratio, and the pancreatic contents of DNA, protein, amylase, lipase, and trypsinogen were measured. A biopsy for histology was taken. Hot water immersion significantly elevated the HSP72 expression, while cold water immersion significantly increased the HSP60 expression. Cold water immersion pretreatment ameliorated the pancreatic edema in trypsin-induced pancreatitis, however this was not due to the HSP60. Hot water immersion pretreatment did not have any effect on the measured parameters in trypsin-induced pancreatitis. The findings suggest that the induction of HSP60 or HSP72 are not enough to protect rats against the early phase of this localized necrohemorrhagic pancreatitis model. Received: 8 October 2001 / Accepted: 26 March 2002     

Elevated levels of antibodies against 70 kDa heat shock proteins in the sera of patients with HIV infection

Heat shock proteins (Hsp), especially 70 kDa heat shock protein (Hsp70) play an important role in the life cycle of HIV-1 virus. Hsp70 is overexpressed in HIV-infected cells and this is the most abundant Hsp associated with HIV virions. The aim of our study was to investigate whether HIV infection increases the extent of specific humoral immune response against Hsp70. The serum concentration of anti-Hsp70 IgG antibodies was measured in 47 HIV-infected patients, and 62 healthy, HIV-seronegative persons. Nineteen patients on highly active anti-retroviral therapy (HAART) were followed for 24 months in a longitudinal study. Anti-Hsp70 antibodies were measured by ELISA, using recombinant human Hsp70. Levels of anti-Hsp70 antibodies were significantly (P < 0.0001) higher in the HIV-infected patients (median: 1409 (25th-75th percentile: 1031-2214) AU/ml) than in healthy control subjects (626 (429-970) AU/ml). In 19 HIV patients, serum levels of anti-Hsp70 antibodies significantly (P < 0.001) decreased during 24 (11-41) months HAART (1309 (887-2213) AU/ml before and 640 (386-959) AU/ml during HAART), accompanied by viral load reduction and CD4+ count elevation. It is concluded that HIV-infection induces a marked increase in the anti-Hsp70 antibody levels, which is consistent with the enhanced expression of Hsp70 on the surface of HIV-infected cells and/or incorporation of the protein into the membrane of HIV virions.