Effect of millimeter waves on cyclophosphamide induced suppression of T cell functions

The effects of low power electromagnetic millimeter waves (MWs) on T cell activation, proliferation, and effector functions were studied in BALB/c mice. These functions are important in T-lymphocyte mediated immune responses. The MW exposure characteristics were: frequency = 42.2 GHz; peak incident power density = 31 +/- 5 mW/cm(2), peak specific absorption rate (SAR) at the skin surface = 622 +/- 100 W/kg; duration 30 min daily for 3 days. MW treatment was applied to the nasal area. The mice were additionally treated with cyclophosphamide (CPA), 100 mg/kg, a commonly used immunosuppressant and anticancer drug. Four groups of animals were used in each experiment: naive control (Naive), CPA treated (CPA), CPA treated and sham exposed (CPA + Sham), and CPA treated and MW exposed (CPA + MW). MW irradiation of CPA treated mice significantly augmented the proliferation recovery process of T cells (splenocytes). A statistically significant difference (P <.05) between CPA and CPA + MW groups was observed when cells were stimulated with an antigen. On the other hand, no statistically significant difference between CPA and CPA-Sham groups was observed. Based on flow cytometry of CD4(+) and CD8(+) T cells, two major classes of T cells, we show that CD4(+) T cells play an important role in the proliferation recovery process. MW exposure restored the CD25 surface activation marker expression in CD4(+) T cells. We next examined the effector function of purified CD4(+) T cells by measuring their cytokine profile. No changes were observed after MW irradiation in interleukin-10 (IL-10) level, a Th2 type cytokine, while the level of interferon-gamma (IFN-gamma), a Th1 type cytokine was increased twofold. Our results indicate that MWs enhance the effector function of CD4(+) T cells preferentially, through initiating a Th1 type of immune response. This was further supported by our observation of a significant enhancement of tumor necrosis factor-alpha (TNF-alpha) production by peritoneal macrophage's in CPA treated mice. The present study shows MWs ameliorate the immunosuppressive effects of CPA by augmenting the proliferation of splenocytes, and altering the activation and effector functions of CD4(+) T cells.     

Effect of millimeter waves on natural killer cell activation

Millimeter wave therapy (MMWT) is being widely used for the treatment of many diseases in Russia and other East European countries. MMWT has been reported to reduce the toxic effects of chemotherapy on the immune system. The present study was undertaken to investigate whether millimeter waves (MMWs) can modulate the effect of cyclophosphamide (CPA), an anticancer drug, on natural killer (NK) cell activity. NK cells play an important role in the antitumor response. MMWs were produced with a Russian-made YAV-1 generator. The device produced modulated 42.2 +/- 0.2 GHz radiation through a 10 x 20 mm rectangular output horn. Mice, restrained in plastic tubes, were irradiated on the nasal area. Peak SAR at the skin surface and peak incident power density were measured as 622 +/- 100 W/kg and 31 +/- 5 mW/cm2, respectively. The maximum temperature elevation, measured at the end of 30 min, was 1 degrees C. The animals, restrained in plastic tubes, were irradiated on the nasal area. CPA injection (100 mg/kg) was given intraperitoneally on the second day of 3-days exposure to MMWs. All the irradiation procedures were performed in a blinded manner. NK cell activation and cytotoxicity were measured after 2, 5, and 7 days following CPA injection. Flow cytometry of NK cells showed that CPA treatment caused a marked enhancement in NK cell activation. The level of CD69 expression, which represents a functional triggering molecule on activated NK cells, was increased in the CPA group at all the time points tested as compared to untreated mice. However, the most enhancement in CD69 expression was observed on day 7. A significant increase in TNF-alpha level was also observed on day 7 following CPA administration. On the other hand, CPA caused a suppression of the cytolytic activity of NK cells. MMW irradiation of the CPA treated groups resulted in further enhancement of CD69 expression on NK cells, as well as in production of TNF-alpha. Furthermore, MMW irradiation restored CPA induced suppression of the cytolytic activity of NK cells. Our results show that MMW irradiation at 42.2 GHz can up-regulate NK cell functions.     

Role of tumor necrosis factor-alpha and interferon-gamma in Helicobacter pylori infection

Immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contributions of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using TNF-alpha geneknockout (TNF-alpha(-/-)) mice and IFN-gamma gene-knockout (IFN-gamma(-/-)) mice. We first examined the colonizing ability of H. pylori strain CPY2052 in the stomach of C57BL/6 wild-type and knockout mice. The number of H. pylori colonized in the stomach of IFN-gamma(-/-) and TNF-alpha(-/-) mice was higher than that of wild-type mice. These findings suggest that TNF-alpha and IFN-gamma may play a protective role in H. pylori infection. Furthermore, we examined the contribution of TNF-alpha and IFN-gamma to gastric inflammation. The CPY2052-infected TNF-alpha(-/-) mice showed a moderate infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium as did wild-type mice, whereas the CPY2052-infected IFN-gamma(-/-) mice showed no inflammatory findings even 6 months after infection. These results demonstrate that IFN-gamma may play an important role in gastric inflammation induced by H. pylori infection, whereas TNF-alpha may not participate in the development of inflammatory response.     

Rat lymphocytes express NMDA receptors that take part in regulation of cytokine production

Incubation of rat lymphocytes with homocysteine (HC) or homocysteic acid (HCA) was found to increase the stationary levels of free radicals in lymphocytes, the effect of both ligands being mediated by ionotropic receptors activated by N-methyl-D-aspactic acid (NMDA), the expression of which on rat lymphocyte membranes was earlier demonstrated. In agreement with these data, increase of free radicals in the lymphocyte cytoplasm is preceded by an increase in the intracellular calcium levels, activation of protein kinase C, nicotinamide adenine dinucleotide phosphate oxidase and/or nitric oxide synthase. Both HC and HCA increase the production of IFN-γ and TNF-α by lymphocytes and antagonist of NMDA receptors; MK-801 prevents this effect. The data presented show that rat lymphocyte membrane contains functionally active NMDA receptors, which regulate cytokine accumulation.     

Effects of different intensities of extremely low frequency pulsed electromagnetic fields on formation of osteoclast-like cells

Over the past 30 years, the beneficial therapeutic effects of selected low energy, time varying electromagnetic fields (EMF) have been documented with increasing frequency to treat therapeutically resistant problems of the musculoskeletal system. However, the underlying mechanisms at a cellular level are still not completely understood. In this study, the effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMF) on osteoclastogenesis, cultured from murine bone marrow cells and stimulated by 1,25(OH)(2)D(3), were examined. Primary bone marrow cells were cultured from mature Wistar rats and exposed to ELF-PEMF stimulation daily for 7 days with different intensities of induced electric field (4.8, 8.7, and 12.2 micro V/cm rms) and stimulation times (0.5, 2, and 8 h/day). Recruitment and authentication of osteoclast-like cells were evaluated, respectively, by determining multinuclear, tartrate resistant acid phosphatase (TRAP) positive cells on day 8 of culture and by the pit formation assay. During the experiments, cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and prostaglandin-E(2) (PGE(2)) were assayed using the enzyme linked immunosorbent assay (ELISA). These findings suggest that ELF-PEMF can both enhance (approximately 50%) and suppress (approximately 27%) the formation of osteoclast-like cells in bone marrow culture, depending on the induced electric field intensity. In addition, consistent correlations were observed between TNF-alpha, IL-1beta, and osteoclast-like cell number after exposure to different induced electric field intensities of ELF-PEMF. This in vitro study could be considered as groundwork for in vivo ELF-PEMF clinical applications on some osteoclast-associated bone diseases.     

Burkholderia pseudomallei interferes with inducible nitric oxide synthase (iNOS) production: a possible mechanism of evading macrophage killing

Burkholderia pseudomallei is a causative agent of melioidosis, a life threatening disease which affects humans and animals in tropical and subtropical areas. This bacterium is known to survive and multiply inside cells such as macrophages. The mechanism of host defense against this bacterium is still unknown. In this study, we demonstrated that B. pseudomallei exhibited unique macrophage activation activity compared with Escherichia coli and Salmonella typhi. The mouse macrophage cell line (RAW 264.7) infected with B. pseudomallei at MOI of 0.1:1, 1:1 and 10:1 did not express a detectable level of inducible nitric oxide synthase (iNOS). Moreover, the B. pseudomallei infected cells released TNF-alpha only when they were infected with high MOI (10:1). Unlike the cells infected with B. pseudomallei, the cells infected with E. coli, and S. typhi expressed iNOS even at MOI of 0.1:1. These infected cells also released a significantly higher level of TNF-alpha at the low MOI ratio. The cells that were preactivated with IFN-gamma prior to being infected with B. pseudomallei exhibited an enhanced production of iNOS and TNF-alpha release. The increased macrophage activation activity in the presence of IFN-gamma also correlated with the restriction of the intracellular bacteria survival. Moreover, IFN-gamma also prevented cell fusion and multinucleated cell formation induced by B. pseudomallei, a phenomenon recently described by our group. Altogether, these results indicate that internalization of B. pseudomallei failed to trigger substantial macrophage activation, a phenomenon which could prolong their survival inside the phagocytic cells and facilitate a direct cell to cell spreading of B. pseudomallei to neighboring cells.     

BH(4) (tetrahydrobiopterin)-dependent activation, but not the expression, of inducible NOS (nitric oxide synthase)-2 in proinflammatory cytokine-stimulated, cultured normal human astrocytes is mediated by MEK-ERK kinases

Nitric oxide (NO) from astrocytes is one of the signalers used by the brain's extensive glial-neuronal-vascular network, but its excessive production by pro-inflammatory cytokine-stimulated glial cells can be cytodestructive. Here, we show how three pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma) together stimulated the activation, but not the prior expression, of NOS-2 protein via a mechanism involving MEK-ERKs protein kinases in astrocytes from adult human cerebral temporal cortex. The cytokines triggered a transient burst of p38 MAPK activity and the production of NOS-2 mRNA which were followed by bursts of MEK-ERK activities, synthesis of the NOS-2 co-factor tetrahydrobiopterin (BH(4)), a build-up of NOS-2 protein and from it active NOS-2 enzyme. Selectively inhibiting MEK1/MEK2, but not the earlier burst of p38 MAPK activity, with a brief exposure to U0126 between 24 and 24.5 h after adding the cytokine triad affected neither NOS-2 expression nor NOS-2 protein accumulation but stopped BH(4) synthesis and the assembly of the NOS-2 protein into active NOS-2 enzyme. The complete blockage of active NOS-2 production by the brief exposure to U0126 was bypassed by simply adding BH(4) to the culture medium. Therefore, this cytokine triad triggered two completely separable, tandem operating mechanisms in normal human astrocytes, the first being NOS-2 gene expression and accumulation of NOS-2 protein and the second being the synthesis of the BH(4) factor needed to dimerize the NOS-2 protein into active, NO-making NOS-2 enzyme.     

Long-term effects of tumor necrosis factor-alpha treatment on insulin signaling pathway in HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB

Tumor necrosis factor-alpha (TNF-alpha) mediated attenuation of insulin signaling pathway is an important cause in several disorders like obesity, obesity linked diabetes mellitus. TNF-alpha actions vary depending upon concentration and time of exposure in various cells. In the present study, the effects of long-term TNF-alpha (1 ng/ml) exposure on the components of insulin signaling pathway in HepG2 and HepG2 cells overexpressing constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB) have been investigated. In parental HepG2 cells, TNF-alpha treatment for 24 h reduced the phosphorylation of Akt1/PKB-alpha and GSK-3beta and under these conditions cells also showed reduced insulin responsiveness in terms of Akt1/PKB-alpha and GSK-3beta phosphorylation. TNF-alpha pre-incubated HepG2-CA-Akt/PKB cells showed lower reduction in Akt1/PKB-alpha and GSK-3beta phosphorylation and insulin responsiveness after 24 h as compared to parental HepG2 cells. We report that the long-term TNF-alpha pre-incubation in both parental HepG2 and HepG2-CA-Akt/PKB-alpha cells leads to the reduction in the levels of IRS-1 without altering the levels of IRS-2. In order to understand the reason for the differential insulin resistance in both the cell types, the effect of long-term TNF-alpha treatment on the proteins upstream to Akt/PKB was investigated. TNF-alpha pre-incubation also showed reduced insulin-stimulated Tyr phosphorylation of insulin receptor (IR-beta) in both the cell types, moreover hyperphosphorylation of IRS-1 at Ser 312 residue was observed in TNF-alpha pre-incubated cells. As hyperphosphorylation of IRS-1 at Ser 312 can induce its degradation, it is possible that reduced insulin responsiveness after long-term TNF-alpha pre-incubation observed in this study is due to the decrease in IRS-1 levels.     

Involvement of 15-lipoxygenase in the inflammatory arthritis

15-Lipoxygenase (15-LOX) is involved in many pathological processes. The aim of this study is to examine the role of 15-LOX in the matrix metalloproteinase (MMP) expression and inflammatory arthritis. It was found that treatment of 15-LOX downstream product of 15-(S)-HETE (15-S-hydroxyeicosatetraenoic acid) increased the mRNA and protein levels of MMP-2 in rheumatoid arthritis synovial fibroblast (RASF) derived from rheumatoid arthritis patients. The enhancement effect of 15-(S)-HETE was antagonized by the addition of LY294002 (PI3K inhibitor) and PDTC (NF-κB inhibitor). Treatment of 15-(S)-HETE increased the phosphorylation of AKT, nuclear translocation of p65 and the breakdown of IκBα. TNF-α and IL-1β are the key cytokines involved in arthritis and also increase the activity of MMP-2 in RASF, which was antagonized by pretreatment with 15-LOX inhibitor PD146176 or knockdown of 15-LOX. It was also found that these two cytokines increased the expression of 15-LOX in RASF. Treatment of glucocorticoid but not NSAIDs inhibited 15-(S)-HETE-induced expression of MMP-2. In comparison with wild-type mice, adjuvant-induced arthritis and MMP-2 expression in synovial membrane were markedly inhibited in 15-LOX knockout (KO) mice. These results indicate that 15-LOX plays an important role in the disease progression of arthritis and may be involved in the inflammatory action induced by TNF-α and IL-1β. 15-LOX is thus a good target for developing drugs in the treatment of inflammatory arthritis.     

A new active vitamin D analog,ED-71, causes increase in bone mass with preferential effects on bone in osteoporotic patients

As a candidate for active vitamin D analogs that have selective effects on bone, 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71) has been synthesized and is currently under clinical trials. In ovariectomized rat model for osteoporosis, ED-71 caused an increase bone mass at the lumbar vertebra to a greater extent than 1alpha-hydroxyvitamin D3 (alfacalcidol), while enhancing calcium absorption and decreasing serum parathyroid hormone levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers.An early phase II clinical trial was conducted with 109 primary osteoporotic patients. The results indicate that oral daily administration of ED-71 (0.25, 0.5, 0.75, and 1.0 microgram) for 6 months increased lumbar bone mineral density in a dose-dependent manner without causing hypercalcemia and hypercalciuria. ED-71 also exhibited a dose-dependent suppression of urinary deoxypyridinoline with no significant reduction in serum osteocalcin. These results demonstrate that ED-71 has preferential effects on bone with diminished effects on intestinal calcium absorption. ED-71 offers potentially a new modality of therapy for osteoporosis with selective effects on bone.     

Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan

Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells into multinuclear cells. TRAP-positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS-induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)-alpha and peptidoglycan (PGN) induced cell fusion, but M-CSF did not. The cell fusion induced by RANKL, TNF-alpha, and LPS was specifically blocked by osteoprotegerin (OPG), anti-TNF-alpha antibody, and polymyxin B, respectively. LPS- and PGN-induced cell fusion was partly inhibited by anti-TNF-alpha antibody but not by OPG. When TRAP-positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal-regulated kinase (ERK), p38MAPK (p38), and c-Jun NH2-terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK-ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP-positive mononuclear cells treated with or without M-CSF, RANKL, TNF-alpha, LPS, or PGN. Collectively, RANKL, TNF-alpha, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC-STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion-inducing factors other than DC-STAMP might be necessary for the cell fusion.     

Effects of millimeter waves on ionic currents of Lymnaea neurons

The effects of mm‐waves 60.22–62.22 GHz and 75 GHz on A‐type K⁺ currents and the effects of 61.22 GHz on Ca²⁺ currents of Lymnaea neurons were investigated using a whole‐cell voltage‐clamp technique. The open end of a rectangular waveguide covered with a thin Teflon film served as a radiator. Specific absorption rates at the waveguide outlet, inserted into physiological solution, were in the range of 0–2400 W/kg. Millimeter wave irradiation increased the peak amplitudes, activation rates, and inactivation rates of both ion currents. The changes in A‐type K⁺ current were not dependent on the irradiation frequency. It was shown that the changes in the amplitudes and kinetics of both currents resulted from the temperature rise produced by irradiation. No additional effects of irradiation on A‐type K⁺ current other than thermal were found when tested at the phase transition temperature or in the presence of ethanol. Ethanol reduced the thermal effect of irradiation. Millimeter waves had no effect on the steady‐state activation and inactivation curves, suggesting that the membrane surface charge and binding of calcium ions to the membrane in the area of channel locations did not change. Bioelectromagnetics 20:24–33, 1999. © 1999 Wiley‐Liss, Inc.     

Caspase cleavage product lacking amino-terminus of IkappaBalpha sensitizes resistant cells to TNF-alpha and TRAIL-induced apoptosis

In response to a diverse array of signals, IkappaBalpha is targeted for phosphorylation-dependent degradation by the proteasome, thereby activating NF-kappaB. Here we demonstrate a role of the cleavage product of IkappaBalpha in various death signals. During apoptosis of NIH3T3, Jurkat, Rat-1, and L929 cells exposed to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas, serum deprivation, or TNF-alpha, respectively, IkappaBalpha was cleaved in a caspase-dependent manner. In vitro and in vivo cleavage assays and site-directed mutagenesis showed that caspase-3 cleaved IkappaBalpha between Asp31 and Ser32. Expression of the cleavage product lacking amino-terminus (1-31), DeltaIkappaBalpha, sensitized otherwise resistant NIH3T3 fibroblast cells to apoptosis induced by TNF-alpha or TRAIL, and HeLa tumor cells to TNF-alpha. DeltaIkappaBalpha was more pro-apoptotic compared to wild type or cleavage-resistant (D31E)IkappaBalpha mutant and the sensitization elicited by DeltaIkappaBalpha was as effective as that by the dominant negative mutant, (S32,36A)IkappaBalpha, in NIH3T3 cells. DeltaIkappaBalpha suppressed the transactivation of NF-kappaB induced by TNF-alpha or TRAIL, as reflected by luciferase-reporter activity. Conversely, expression of the p65 subunit of NF-kappaB suppressed TNF-alpha-, TRAIL-, and serum deprivation-induced cell death. On the contrary, DeltaIkappaBalpha was less effective at increasing the death rate of HeLa cells that were already sensitive to death signals including TRAIL, etoposide, or taxol. These results suggest that DeltaIkappaBalpha generated by various death signals sensitizes cells to apoptosis by suppressing NF-kappaB activity.     

Phytoestrogens directly inhibit TNF-α-induced bone resorption in RAW264.7 cells by suppressing c-fos-induced NFATc1 expression

TNF-α-induced osteoclastogenesis is central to post-menopausal and inflammatory bone loss, however, the effect of phytoestrogens on TNF-α-induced bone resorption has not been studied. The phytoestrogens genistein, daidzein, and coumestrol directly suppressed TNF-α-induced osteoclastogenesis and bone resorption. TRAP positive osteoclast formation and resorption area were significantly reduced by genistein (10(-7) M), daidzein (10(-5) M), and coumestrol (10(-7) M), which was prevented by the estrogen antagonist ICI 182,780. TRAP expression in mature TNF-α-induced osteoclasts was also significantly reduced by these phytoestrogen concentrations. In addition, in the presence of ICI 182,780 genistein and coumestrol (10(-5) -10(-6) M) augmented TNF-α-induced osteoclast formation and resorption. However, this effect was not observed in the absence of estrogen antagonist indicating that genistein's and coumestrol's ER-dependent anti-osteoclastic action normally negates this pro-osteoclastic effect. To determine the mechanism mediating the anti-osteoclastic action we examined the effect of genistein, coumestrol, and daidzein on caspase 3/7 activity, cell viability and expression of key genes regulating osteoclast differentiation and fusion. While anti-osteoclastic phytoestrogen concentrations had no effect on caspase 3/7 activity or cell viability they did significantly reduce TNF-α-induced c-fos and NFATc1 expression in an ER dependent manner and also inhibited NFATc1 nuclear translocation. Significant decreases in NFκB and DC-STAMP levels were also noted. Interestingly, constitutive c-fos expression prevented the anti-osteoclastic action of phytoestrogens on differentiation, resorption and NFATc1. This suggests that phytoestrogens suppress TNF-α-induced osteoclastogenesis via inhibition of c-fos-dependent NFATc1 expression. Our data provides further evidence that phytoestrogens have a potential role in the treatment of post-menopausal and inflammatory bone loss directly inhibiting TNF-α-induced resorption.     

Ultraviolet B irradiation modulates susceptibility to tumour necrosis factor-alpha-induced apoptosis via induction of death receptors in murine fibroblasts.

Ultraviolet B (UVB) irradiation causes cell death by apoptosis in murine fibroblast cells. Tumor necrosis factor-alpha (TNF-alpha) is also a well known inducer of apoptosis, although the physiological significance of this activity is poorly understood. We investigated the effects of pretreatment with UVB (312 nm) on TNF-alpha-induced apoptosis in murine fibroblast cells. UVB enhanced susceptibility to cell death by TNF-alpha in a dose-dependent manner. UVB but not TNF-alpha induced the expression of TNF receptor type-1 (TNFR-1) and type-2 (TNFR-2) in a dose-dependent manner. Expression of Fas (CD95) and Fas-ligand (Fas-L), and significant DNA fragmentation were observed in the cells that died. These results suggest that UVB irradiation modulates susceptibility to TNF-alpha-induced apoptosis through the induction of TNFRs, Fas, and Fas-L in murine fibroblasts.     

High-dose corticosteroid administration induces increase of serum macrophage migration inhibitory factor in patients with Vogt-Koyanagi-Harada's disease

To investigate the influence of corticosteroid administration on the serum level of macrophage migration inhibitory factor (MIF), sera obtained from 9 patients with Vogt-Koyanagi-Harada's disease who had been treated with high-dose corticosteroid were analyzed. The serum MIF levels of most patients were prominently increased on day 7 and/or day 14 after corticosteroid treatment. No TNF-alpha was detected in the sera. The average serum MIF level of nine patients at the highest stages after corticosteroid administration was significantly higher than that before the corticosteroid treatment. It seems that MIF is a unique cytokine and acts together with corticosteroid to regulate inflammation and immunity.