Interleukin-1 (IL-1) receptor antagonist prevents Staphylococcus epidermidis-induced hypotension and reduces circulating levels of tumor necrosis factor and IL-1 beta in rabbits.

Similar to shock in gram-negative sepsis, shock from gram-positive organisms is mediated, in part, by tumor necrosis factor (TNF) and interleukin-1 (IL-1). In the present study, rabbits were infused with IL-1 receptor antagonist (IL-1ra) prior to and during Staphylococcus epidermidis-induced hypotension. After injection of bacteria, a maximal fall in mean arterial pressure to -42% below baseline occurred at 200 min in vehicle-treated animals compared with a nonsignificant decrease of only 7% in the IL-1ra-treated group (P < 0.01, vehicle versus IL-1ra). A similar attenuation was observed in the fall in systemic vascular resistance (P < 0.05). After the injection of S. epidermidis, TNF levels rose to a peak elevation of 475 +/- 160 U/ml in vehicle-treated rabbits, but in rabbits receiving IL-1ra, maximal TNF levels rose only to 85 +/- 23 U/ml (P < 0.01). Plasma IL-1 beta reached maximal concentrations at 180 min of 364 +/- 71 pg/ml in vehicle-treated animals but only 145 +/- 12 pg/ml in rabbits given IL-1ra (P < 0.05). The reductions in TNF and IL-1 were not due to interference by IL-1ra in the respective assays. In vitro, IL-1ra inhibited S. epidermidis-induced TNF from mononuclear cells by 31% +/- 11%, from spleen cells by 17% +/- 4% (P < 0.05), and from whole blood by 42% +/- 17%. Despite the near reversal of the fall in mean arterial pressure and systemic vascular resistance in IL-1ra-treated rabbits, leukopenia and thrombocytopenia were unaffected. These results demonstrate that IL-1ra blocks shock-like hemodynamic parameters and reduces circulating IL-1 and TNF levels in a model of gram-positive sepsis.     

Cytokines in symptomatic asthma airways.

To determine whether cytokines are generated in vivo in subjects with asthma, we have measured cytokine levels (tumor necrosis factor [TNF], granulocyte-macrophage-colony-stimulating factor [GM-CSF], interleukin [IL]-1 alpha, IL-1 beta, IL-2, IL-4, and IL-6) in the airways of subjects with symptomatic (N = 24) and asymptomatic (N = 9) asthma with immunoassays (GM-CSF, IL-1 alpha, IL-1 beta, IL-2, and IL-4) or bioassays (TNF and IL-6) and the polymerase chain reaction (IL-1 beta and TNF). Significant levels of TNF (578 +/- 917 pg/ml versus 24 +/- 29 pg/ml) (p = 0.01), GM-CSF (24 +/- 41 pg/ml versus less than 8 pg/ml) (p = 0.02), and IL-6 (225 +/- 327 pg/ml versus 7 +/- 12 pg/ml) (p = 0.01), but not IL-1 alpha or IL-4, were detected in the bronchoalveolar lavage fluid (BALF) of patients with symptomatic compared with BALF of patients with asymptomatic asthma. Levels of IL-1 beta (266 +/- 270 pg/ml versus less than 20 pg/ml) (p = 0.001) and IL-2 (1.4 +/- 2.8 ng/ml versus less than 0.3 ng/ml) (p = 0.05) in BALF in patients with symptomatic compared with that in BALF levels in patients with asymptomatic asthma suggested activation of alveolar macrophages and T cells. Thus, in episodes of asthma, several cytokines, including TNF, GM-CSF, IL-1 beta, IL-2, and IL-6 are detectable in BALF.     

Production of tumor necrosis factor-alpha and interleukin-6 by human alveolar macrophages exposed in vitro to coal mine dust.

Following our previous demonstration of cytokine secretion by alveolar macrophages (AM) from coal miners and from patients with coal workers' pneumoconiosis, we investigated the effect of in vitro exposure to coal dust and to its silica content on tumor necrosis factor-alpha (TNF), interleukin (IL)-1 beta, and IL-6 production by normal human AM. TNF and IL-1 beta concentrations were estimated by a specific radioimmunoassay, while IL-6 levels were evaluated by the proliferation of 7TD1 cells. After 24-h culture, coal dust triggered a significant release of TNF and IL-6 at the dose of 0.1 mg/ml and more obviously at 1 mg/ml in comparison with titanium dioxide (TiO2), used as a biologically inert control dust (with 1 mg/ml of dust: 3,526 +/- 3,509 versus 330 +/- 138 pg TNF/ml and 224 +/- 74 versus 72 +/- 34 U IL-6/ml, respectively; P less than 0.01 in both cases). After 3-h culture, a significant TNF secretion as well as an increased TNF mRNA expression were also detected for AM stimulated by coal dust at variance with TiO2. In contrast, no modification of IL-1 beta concentration could be evidenced in AM exposed to coal dust, although we detected an increased expression of specific mRNA expression. In order to define the role of silica among the main components of coal dust in AM activation, we evaluated the effect of silica (alpha-quartz, 30 micrograms/ml, which is the concentration and the type of silica present in our coal dust) alone or mixed with TiO2 (1 mg/ml) on monokine production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-activating factor modulates interleukin-6 production by mouse fibroblasts

We compared the effects of platelet-activating factor (PAF), interleukin-1 beta (IL-1 beta) and polyriboinositic-polyribocytidylic acid (poly-I:C) on IL-6 production by confluent L929 mouse fibroblasts. At concentrations above 1 microM, PAF dose-dependently enhanced IL-6 production; at 5 microM PAF this increase (72.7 +/- 19.9 U/ml) was higher than that evoked by 100 U/ml IL-1 beta (5.7 +/- 0.4 U/ml) or 50 micrograms/ml poly-I:C (39.3 +/- 6.7 U/ml). The IL-6 production induced by 5 microM PAF was not inhibited by addition of the specific PAF antagonist BN 52021 (10 microM) to the incubation medium. These results demonstrate that, as this is the case for IL-1, PAF also modulates IL-6 production.     

Association between HLA-DR2 and Production of Tumour Necrosis Factor α and Interleukin 1 by Mononuclear Cells Activated by Lipopolysaccharide

The production of tumour necrosis factor (TNF) and interleukin 1 (IL-1) by lipopolysaccharide-activated mononuclear cells from 39 healthy donors was studied in vitro by bioassay and ELISA. The donors were typed for HLA-A, -B, -C, -DR, and -DP antigens. There was no detectable production of TNF beta (lymphotoxin). The intracellular levels of bioactive TNF alpha were minimal or undetectable in all cases. Cells from HLA-DR2+ individuals secreted significantly lower amounts of TNF alpha than cells from HLA-DR2- donors [2 ng/ml (1.5-4.4) and 7.5 ng/ml (3.9-8.3) respectively (medians 25-75%); P less than 0.01]. The difference disappeared if the cells were preactivated for 2 days with 1000 U/ml of recombinant gamma interferon (rIFN-gamma). In some individuals, the TNF alpha response increased considerably after IFN-gamma priming, in particular in those possessing the HLA-DR2 antigen. In contrast, there was no detectable difference in the production of IL-1 beta between the donors, and the IL-1 beta response decreased significantly after rIFN-gamma priming in HLA-DR2+ individuals [2.3 ng/ml (1.1-8.4) versus 7.2 ng/ml (5-7.9); P less than 0.05] and in HLA-DR2- individuals [3 ng/ml (1.1-5.3) versus 5.7 ng/ml (3.9-7.5); P less than 0.01]. There was no correlation between the TNF alpha and IL-1 responses and any of the other HLA-DR, -DP, or -B antigens. There was a significant positive correlation between the levels of TNF alpha measured by ELISA and by cytotoxicity assay. However, the TNF alpha-containing supernatants from 9 out of 37 individuals appeared to contain inhibitor(s) of the biological activity of TNF alpha. The presence of inhibitor(s) was not associated with any HLA antigens.     

OK-432-induced cytokines production by peripheral blood mononuclear cells]

Cytokines production by OK-432-stimulated peripheral blood mononuclear cells (PBMC) were measured to investigate the in vitro function of macrophages (M phi) and lymphocytes. PBMC (1 x 10(6) cells/ml) were cultured with OK-432 (0.05 KE/ml) for 72 hr at 37 degrees C under 5% CO2, then interleukin 1 beta (IL-1 beta), interleukin 2 (IL-2) and soluble IL-2 receptor (sIL-2R) levels in the culture supernatants were measured by ELISA. While there was no significant differences of IL-1 beta production between patients with chronic active hepatitis type B (CAH-B) and controls, sIL-2R production (335 +/- 219 U/ml, mean +/- SD) was significantly decreased (p < 0.001) in patients with CAH-B. On the other hand, in pregnant women, production of both IL-1 beta (6.3 +/- 3.9 ng/ml, p < 0.01) and sIL-2R (300 +/- 169 U/ml, p < 0.001) were significantly lower than those in controls (13.5 +/- 3.8 ng/ml, 969 +/- 154 U/ml). These results suggest that the expression of IL-2R alpha on lymphocytes membrane is suppressed in patients with CAH-B, and that decreased M phi function is present in pregnant women.     

Interleukin-6 production by tumor necrosis factor and lipopolysaccharide-stimulated rat renal cells

Interleukin-6 (IL-6) is produced by various cell types, including monocytes, fibroblasts, and endothelial cells. IL-6 has also been detected in the urine of normal and renal transplant patients. Thus, the possible production of this cytokine by glomeruli and mesangial cells was investigated. Rat glomeruli were obtained by serial sieving of cortical homogenates of blood-free kidneys. Mesangial cells were obtained from the glomeruli and cultured under standard methods in RPMI 1640 medium containing 15% fetal calf serum. Glomeruli or confluent monolayers cells were then incubated in RPMI 1640 for 18 hr, in the presence or not of tumor necrosis factor-alpha (TNF alpha), lipopolysaccharide (LPS), or platelet-activating factor (PAF). IL-6 activity was measured using the IL-6-dependent cell line subclone (B 9-9) and expressed with respect to a standard curve established with recombinant IL-6. Glomeruli generate IL-6 upon TNF alpha (100 ng/ml) and LPS (1 microgram/ml), 11,500 +/- 3000 and 22,000 +/- 7500 U/ml, respectively. Nonstimulated mesangial cells produced 50 +/- 5 U/ml (mean +/- SEM, n = 4) of IL-6. TNF alpha (1 ng/ml) and LPS (1 microgram/ml) induced the production of 800 +/- 90 and 40,000 +/- 5000 U/ml, respectively (n = 4). In contrast, PAF (0.1 nM-1 microM) did not increase IL-6 production from glomeruli or mesangial cells. These results demonstrate that renal cells spontaneously generate minimal amounts of IL-6 and that this production is significantly increased by TNF alpha or LPS. A synergy between LPS and TNF alpha was induced in glomerular cells with 10 ng/ml of TNF alpha and graded concentrations of LPS. Thus, the production of IL-6 by glomerular cells and its modulation by other cytokines or endotoxins may play a role in the local immunological processes leading to immune glomerular diseases.     

Platelet activation markers and soluble adhesion molecules in patients with systemic lupus erythematosus

We assessed the role of platelet activation markers (PMPs, Annexin V and CD62P on activated platelets), cytokines (IL-1 beta, IL-4, IL-6, IFN- gamma, GM-CSF, and TNF alpha ), and soluble factors (sIL-2R, TM, sHLA-1, beta(2) -m, sVCAM-1, sPECAM-1, sP-selectin and sE-selectin) in vascular damage related to SLE. There were differences in the levels of PMPs and platelet activation markers between the SLE patients and controls (PMPs: 493+/-82 vs. 328+/-36, p<0.05; plt-CD62P; 8.5%+/-1.2 % vs. 4.6%+/-0.7 %, p<0.05; plt-Annexin V: 11.3%+/-2.1 % vs. 4.9%+/-0.6 %, p<0.01). There were no differences in the levels of IFN- gamma between the groups. However, the levels of IL-1 beta, IL-4, IL-6, GM-CSF, TNF alpha, and soluble factors were higher in the SLE patients than in the controls. The levels of IL-4, IL-6, beta2 -m, sIL-2R, sVCAM-1, sP-selectin, and sE-selectin in SLE patients with elevated sTM levels were higher than those in the SLE patients without elevated sTM levels. On the other hand, elevations of sIL-2R, sVCAM-1, and sP-selectin were not found in patients with Beh?et disease or rheumatoid arthritis. The levels of platelet CD62P, platelet annexin V, and PMP were significantly elevated in high-sTM patients. These findings suggest the possibility that activated platelets and cytokines participate in the pathogenesis of SLE in patients with elevated sTM levels.     

Cytokine production and serum levels in systemic sclerosis.

Serum levels of various cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL1-beta), and interleukin 2 (IL2), and of soluble IL2 receptors (sIL2R) were determined in 30 patients with definite systemic sclerosis (SSc). Spontaneous and lipopolysaccharide-or mitogen-induced production of the cytokines, TNF-alpha, IL1-beta, and IFN-gamma, by peripheral blood mononuclear cells (PBMNC) of these SSc patients was measured by immunoassays. The patients were divided into three groups: 12 with limited cutaneous disease (lcSSc), 7 with diffuse cutaneous disease (dcSSc) < 3 years duration, and 11 with dcSSc > 3 years duration. None were treated with cytotoxic drugs or biologic response modifiers. Sera of patients with SSc had elevated sIL2R levels, and only low levels of IL2 (1-2 U/ml) were detected in 10/29 sera tested. Spontaneous production of TNF-alpha and IL1-beta by PBMNC of patients with SSc (829 pg/ml +/- 215 SEM and 728 pg/ml +/- 186, respectively) was significantly higher than that by normal PBMNC obtained from 30 volunteers (25 +/- 10 and 34 +/- 6 pg/ml, respectively) and tested at the same time as patients' PBMNC. The largest increases in spontaneous release of TNF-alpha or IL1-beta were seen in patients with early dcSSc. No significant difference in spontaneous IFN-gamma production by patient or control PBMNC was detected. On the other hand, the mean level of mitogen-induced IFN-gamma production by PBMNC was significantly depressed in patients with SSc (103 U/ml +/- 18 vs 255 +/- 33 U/ml in controls). In vitro-induced production of TNF-alpha or IL1-beta by patients' PBMNC was comparable to that of normal PBMNC. These data indicate that in vivo-activated PBMNC of patients with SSc spontaneously secrete excessive amounts of fibrogenic cytokines, which are involved in modulation of connective tissue synthesis.     

Combinations of low concentrations of cytokines and acute agonists synergize in increasing the permeability of endothelial monolayers.

The deposition of circulating immune reactants in blood vessels, an important event in the pathogenesis of certain types of vasculitis, requires an increase in permeability in the endothelial monolayer. An in vitro model to examine the integrity of endothelial cell monolayers and their response to inflammatory mediators has been developed. Human umbilical vein endothelial cells were grown to confluence on an FITC-labelled matrix and monolayer integrity was assessed by the exclusion of a 125I-anti-FITC antibody. Alteration in endothelial monolayer permeability was associated with an increase in uptake of 125I-anti-FITC antibody, expressed as a percentage of the maximal uptake of antibody on to FITC-matrix from which endothelial cells had been stripped. We determined the effects on endothelial monolayer permeability of acute agonists (thrombin and histamine), cytokines (tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-1 and IL-4) and combinations of acute agonists and cytokines. Addition of thrombin in concentrations ranging from 0.5 to 15 U/ml led to an increased uptake of 125I-anti-FITC antibody from 2% to 15% relative to unstimulated endothelium. For other agonists and cytokines the increases in permeability were: (i) histamine (50-400 pmol/ml) increased uptake 5-22%; (ii) TNF (12.5-100 ng/ml) increased uptake 2-12%; (iii) IFN-gamma (125-250 U/ml) increased uptake 1.5-3%. IL-1 beta (50-100 U/ml) and IL-4 (50-100 U/ml) had no effect. Synergistic interactions on endothelial monolayer permeability were seen with the following combinations: (i) IL-4 (100 U/ml) and TNF (12.5 ng/ml) uptake 11%; (ii) IL-4 (100 U/ml) and IFN-gamma (125 U/ml) uptake 6.5%; (iii) TNF (12.5 ng/ml) and IFN-gamma (125 ng/ml) uptake 7%; (iv) thrombin (0.5 U/ml) and histamine (50 pmol/ml) uptake 13.5%; and (v) TNF (12.5 ng/ml) and thrombin (0.5 U/ml) uptake 8.5%. These observations suggest that interactions between cytokines and acute inflammatory mediators such as thrombin and histamine may be important in determining whether immune complexes are deposited in vessel walls. This model system may now be useful for the further investigation in vitro of the mechanisms involved in the pathogenesis of immune complex-mediated vascular damage.     

Detection of Tumour Necrosis Factor from Lipopolysaccharide-Stimulated Human Mononuclear Cells by Enzyme-Linked Immunosorbent Assay and Cytotoxicity Bioassay

Tumour necrosis factor (TNF) or cachectin is an important mediator of endotoxic activity. To investigate the production of TNF from human mononuclear cells (MNC) in response to lipopolysaccharide (LPS), we developed a sensitive and specific enzyme immunoassay (ELISA) and a cytotoxicity bioassay for TNF. The ELISA utilizes the biotin/avidin system and includes four incubation steps. The detection limit was 25 pg recombinant TNF (rTNF)/100 microliter. There was no interference of medium, serum, plasma, spinal fluid, or urine and no cross-reaction with natural or recombinant IL-1-alpha, IL-1-beta, IL-2, IFN-gamma, or lymphotoxin (TNF-beta). Recovery of TNF added to the media was 85-123% (n = 22). The relative standard deviations within and between assays were 7% and 8%, respectively. TNF-induced cytotoxicity was measured on actinomycin-D-treated L-M mouse fibroblasts. The detection limit in this bioassay was 0.5 U/30 microliter or 12.5 pg/30 microliter of rTNF. IL-1-alpha and IL-1-beta slightly inhibited the cytotoxic activity of rTNF. In this bioassay, cytotoxic activity (50-300 U/ml) was detected only when MNC were stimulated with high concentrations of LPS (100-1000 ng/ml). In contrast, using 0.01-100 ng/ml of LPS, the ELISA detected TNF in a dose-dependent manner (0.25 ng/ml to 40 ng/ml). It is concluded that TNF is liberated from human blood MNC if stimulated with minute amounts of LPS. It is suggested that human TNF may be secreted in a relatively inactive form or that inhibitors of TNF are generated along with the monokine. Because of this, and because commonly used bioassays for TNF fail to distinguish between TNF and lymphotoxin, specific ELISA are recommended to supplement TNF bioassays.     

Biocompatibility of high-flux membranes.

Standard dialysis with cuprophane membranes is known to stimulate the immune system. As a result of activation of macrophages various interleukins and tumor necrosis factor (TNF) are secreted, presenting further evidence of the poor biocompatibility of cuprophane. We investigated the immunogenic properties of three modern high-flux membranes. Seven patients were studied during hemodiafiltration sessions using either a polysulfone (F60, Fresenius), a polymethylmetacrylate (BK 2.1, Toray) or a cellulose triacetate (FB-210 U, Nipro) dialyzer in a hemodiafiltration procedure. Serial measurements were made during each treatment of interleukin-1 beta (II-1 beta), TNF, soluble IL-2 receptor (sII-2r), soluble CD4 (sCD4), soluble CD8 (sCD8), interferon gamma (IFNg) and neopterin. In contrast to the known increase of IL-1 beta, IL-2r and TNF with cuprophane membranes, none of the modern high-flux dialyzers stimulated the production of these factors. Significant decreases of neopterin and sCD4 were observed. IFNg and sCD8 did not change significantly. Our results suggest that the modern high-flux dialyzers are non-immunogenic, and thus provide further evidence of the superior biocompatibility of synthetic or semisynthetic membranes over the conventional cuprophane.     

大骨节病患者血清促炎症细胞因子水平的检测

目的探讨前炎症细胞因子TNF、IL-1β和IL-6在大骨节病(KBD)发病机制中的作用。方法采集62例KBD患者和60例健康对照的血清标本,采用双抗体夹心ELISA法测定血清前炎症细胞因子TNF、IL-1β和IL-6的水平。结果KBD患者血清IL-1β和IL-6的水平分别为(238.4±698.5)ng/L和(164.4±661.4)ng/L,健康人分别为(74.5±130.0)ng/L和(52.2±154.6)ng/L,但它们之间差异均不显著(P>0.05)。然而,KBD患者血清TNF的水平[(109.2±145.3)ng/L]高于健康对照[(40.9±89.7)ng/L],差异非常显著(P<0.01)。患者血清TNF与IL-1β的水平及血清TNF与IL-6水平的相关性均不显著(r值分别为0.0387和0.2135,P>0.05)。血清IL-1β与IL-6的水平呈显著的正相关(r=0.3460,P<0.01)。结论血清前炎症细胞因子水平的升高,可能与大骨节病的发病有关。     

Cytokines in patients with lung cancer

Lung cancer is one of the most common malignant diseases and is amongst the leading causes of death. Cell-mediated immune response and cytokines could play an important role in antitumour immunity. The aim of the study was to evaluate the cytokines', tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and IL-6, releasing capacity in patients with lung carcinoma and benign lung disease. A group of 41 patients were tested for the production of TNF-alpha, IL-1beta and IL-6 in bronchoalveolar lavage (BAL) and blood. The levels of cytokines in the lung cancer patients were: (1) in BAL - IL-6, 173 +/- 85 pg/ml; TNF-alpha, 170 +/- 116 pg/ml; and IL-1beta, 473 +/- 440 pg/ml; (2) in the blood - IL-6, 197 +/- 53 pg/ml; TNF-alpha, 311 +/- 202 pg/ml; and IL-1beta, 915 +/- 239 pg/ml. Alveolar macrophages of the patients with a lung cancer secreted significantly more cytokines, IL-6 (P = 0.0004) and IL-1beta (P = 0.0047), than alveolar macrophages of the patients with a nonmalignant lung cancer. However, significantly lower levels of cytokine production by the BAL cells were found in patients with small cell lung cancer. This production decreased further in phase IV of nonsmall cell lung cancer.     

Increase of serum interleukin 2 receptor level in thermally injured patients

Serum concentrations of receptor for the T cell growth factor interleukin 2 (IL-2R) were compared to the capacity of activated T cells to express surface IL-2R in patients with major burns. In immunosuppressed patients the numbers of cells expressing IL-2R were transiently (survivors) or permanently (nonsurvivors) reduced (up to 50 and 90%, respectively). In contrast, the levels of soluble IL-2R in patients' sera were significantly (P less than 0.001-0.05) elevated throughout the postburn period. Within 24 hr postinjury, over 90% of patients demonstrated 400-3500 U/ml of serum IL-2R compared with 120-340 U/ml in normal controls. Soluble IL-2R, when patients became immunosuppressed, further increased to 4445 +/- 962 U/ml in nonsurvivors and to 2031 +/- 578 U/ml in survivors, and demonstrated a significant capacity to inhibit the activity of exogenous IL-2. In survivors the levels of serum IL-2R declined at discharge, to 1009 +/- 104 U/ml. Thus, in thermally injured patients, soluble IL-2 receptor concentrations are elevated, and could interfere with IL-2-mediated immune interactions.     

Switch from cyclosporine A to tacrolimus in renal transplant recipients: impact on Th1, Th2, and monokine responses

We showed previously that pretransplant CD4 helper defects and low in-vitro IL-10 responses predict a low risk of acute kidney graft rejection. To compare the effect of tacrolimus (Tacr) and cyclosporine A (CsA) on the humoral immune response we assessed T helper function, B cell/monocyte responses and in-vitro cytokine responses (TNF-alpha, GM-CSF, IL-1 beta, IL-2, IL-4, IL-6, IL-10) in 20 renal transplant recipients before and 3 months after they were switched from CsA to Tacr because of hyperlipoproteinemia, hirsutism, or gum hyperplasia. T helper function was assessed using a PWM-driven allogeneic coculture system of patient T cells together with control B cells. B cell/monocyte responses were determined using a PWM-stimulated allogeneic coculture system, SAC I-stimulated B-cell cultures and LPS-stimulated monocyte cultures. Immunoglobulin-secreting cell (ISC) responses were assessed in a reverse hemolytic plaque assay, and ELISA were used to determine cytokine secretion. Treatment with Tacr resulted in a decreased expression of costimulatory ligands and adhesion molecules (T cells: CD40L, p < 0.05; CD28 and CD54, p < or = 0.01; B cells: CD25, p = 0.05; CD40, p < 0.001; monocytes: CD40, p < 0.05), which coincided with decreased PHA-stimulated T cell IL-2 responses (398 +/- 153 versus 43 +/- 15 pg/ml, p < 0.05), impaired CD4 helper activity (117% +/- 22% versus 73% +/- 19%, p < 0.05) and increased CD4 suppressor activity (-120% +/- 28% versus -18% +/- 27%, p = 0.02). We observed enhanced CD4 IL-10 responses (p < 0.01) and LPS-stimulated monocyte responses (TNF-alpha, IL-1 beta, and IL-6, p < 0.005; IL-10, p < 0.05), indicating an increased humoral immune responsiveness under treatment with tacrolimus. Our data show that switching of immunosuppressive therapy from CsA to tacrolimus results in suppression of costimulatory ligands, adhesion molecules, Th1 responses and CD4 helper activity. However, enhanced humoral immune responses, Th2 and monokine responses, might have a negative impact on long-term graft function.     

T helper/inducer (CD4+) cells prestimulated with PPD induce monocytes to produce interleukin-1 beta.

We obtained peripheral blood mononuclear cells (PBMC) from four healthy, tuberculin purified protein derivative (PPD) reactive donors and cultured these cells in media containing PPD (low dose = 200 ng/ml or high dose = 1 micrograms/ml). Five days after the addition of PPD, T cells were isolated, washed, and added to autologous adherent cell cultures at a 1:1 ratio. Adherent cells were then cultured for 24 h in media only (baseline), media plus lipopolysaccharide (LPS, 2 micrograms/ml; positive control), or media containing the prestimulated T cells. After 24 h, supernatants were harvested and interleukin 1 beta (IL-beta) levels were assayed by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The results show that T cells prestimulated with low dose PPD (200 micrograms/ml) did not induce IL-1 production by adherent cells (mean increase over baseline 0.2 +/- 1.3 standard deviation [SD] ng/ml, P = 0.61). However, T cells prestimulated with high dose PPD (1 microgram/ml) did induce adherent cells to secrete IL-1 beta (mean increase over baseline 1.7 +/- 0.62 [SD] ng/ml, P = 0.01), but this induction was abolished when cell-to-cell contact was prevented by use of double well chambers (mean increase over baseline 0.1 +/- 0.36 [SD] ng/ml, P = 0.69). Prestimulated T helper (CD4+) cells were able to induce monocytes to secrete IL-1 beta but prestimulated CD8+ T cells were not. These data suggest that when T helper (CD4+) cells are sufficiently activated they acquire the ability to induce monocytes to secrete IL-1 beta. Cell-to-cell contact between monocytes and T cells is required. This function of activated T cells may be important in the normal cellular immune response.     

Protein A of Staphylococcus aureus evokes Th1 type response in mice.

Protein A (PA) of Staphylococcus aureus is known to elicit several cytokines such as IFN gamma, TNF alpha and IL1. However, it has not been delineated yet as to which differentiation pathway lymphocytes follow after stimulation by PA. In this report, we attempted to collect such evidences. Cytokines, such as IFN gamma, IL2, IL4, IL6, IL10, TNF alpha, IL1alpha and IL1beta were measured in serum by ELISA. Our results show that 1 microg dose of PA stimulates the production of IFN gamma (115 +/- 5 pg/ml), TNF alpha (250 +/- 8 pg/ml) and IL1alpha (100 +/- 5 pg/ml) as compared to control levels of, 22 +/- 2, 20 +/- 2 and 35 +/- 3 pg/ml respectively whereas IL2 and IL1beta secretion were less (beyond the lower detection limit of the kit and 25 +/- 1 pg/ml, respectively) as compared to control (28 +/- 2 and 52 +/- 4 pg/ml, respectively). Larger dose of PA (10 microg) increases the expression of IL2 (75 +/- 3 pg/ml), TNF alpha (1380 +/- 120 pg/ml), IL1alpha (495 +/- 10 pg/ml) and IL1beta (110 +/- 7 pg/ml) as compared to controls described above. We also observed that 1 microg dose of PA decreases IL4, IL6 and IL10 secretion to 9 +/- 1, 10 +/- 1 and 10 +/- 2 pg/ml, respectively, whereas 10 microg dose also decreased them to 11 +/- 1, 12 +/- 2 and 30 +/- 4 pg/ml, respectively as compared to the background controls, i.e. 50 +/- 5, 50 +/- 2 and 215 +/- 9 pg/ml respectively. The ratio of IFN gamma to IL4 increased and the peak value at 4 h, came to 13 +/- 1 and 9.6 +/- 0.5 with 1 microg and 10 microg PA, respectively, which is an established parameter indicating a Th1 type response. Flow cytometry analysis of CD4+/CD8+ cells, and c-myc protein expression by splenocytes indicate that 1 microg dose of PA causes 2-fold increase of CD4+ cells with no change in CD8+ cells, and 10-fold increase in c-myc protein, whereas 10 microg dose increases CD4+ cells 4-fold, CD8+ cells 3-fold and c-myc protein 100-fold. The cell cycle data shows an induction of apoptosis in thymocytes and splenocytes with the large dose (10 microg), whereas the 1 microg dose does not show any apoptosis. This report indicates that a Th1 response is induced in mice, after PA inoculation at a dose of 1 microg animal. Thus, cytokine mediated therapeutic strategies should consider the fact that an induction of large concentration of some cytokines might become detrimental to the host.     

Soluble IL-2 receptor and tumour necrosis factor-alpha in plasma of haemophilia patients infected with HIV.

We measured plasma concentrations of soluble receptors for IL-2 (sIL-2R) and tumour necrosis factor-alpha (TNF-alpha) in 149 haemophilia patients. Soluble IL-2R levels were elevated in 37% of 62 HIV-seronegative patients (mean 570 +/- 27 U/ml versus 361 +/- 17 U/ml in the control group, P less than 0.0001), in 78% of 68 HIV-seropositive patients (928 +/- 49 U/ml, P less than 0.0001), and in 95% of 19 AIDS/ARC patients (1578 +/- 199 U/ml, P less than 0.0001 compared with controls and with HIV-seronegative patients; P less than 0.005 compared with HIV-seropositive asymptomatic patients). A negative correlation was observed between sIL-2R, relative and absolute numbers of CD4+ cells (P less than 0.0001), and CD4/CD8 ratios (P less than 0.0001). There was also a negative correlation between sIL-2R in plasma and the cellular expression of IL-2R (P less than 0.001). We found a significant association of sIL-2R and plasma neopterin (P less than 0.0001). With progression of the disease from HIV-seronegative to seropositive without symptoms and to full manifestation of AIDS/ARC, sIL-2R plasma levels increased. The highest levels were found at the time of diagnosis of AIDS/ARC, but the levels decreased again during the following 18 months. Eight per cent of HIV-seronegative patients, 32% of HIV-seropositive patients, and 24% of patients with AIDS/ARC had increased plasma TNF-alpha. We conclude that sIL-2R and TNF-alpha plasma levels are elevated in HIV-infected haemophilia patients and that sIL-2R is a marker for disease progression from asymptomatic HIV-seropositive to AIDS/ARC.     

Effect of immune cytokines on bone

The effect of the bone resorptive cytokines IL-1 alpha, IL-1 beta, and TNF on bone formation was studied in an in vitro system. All three cytokines were profoundly inhibitory, with the rank order of potency IL-1 beta greater than IL-1 alpha greater than TNF. Inhibition was mediated by a depression of differentiated osteoblast functions, including alkaline phosphatase expression and matrix synthesis. Osteoblast proliferation was not affected. Bone formation inhibition was independent of PGE2 production, indicating a direct effect of cytokines on osteoblasts. High concentrations of IL-1 beta (10 U/ml) abrogated IGF-1-stimulated bone formation, providing evidence for the hypothesis that cytokines act as 'uncoupling factors'. Conversely, high concentrations of IGF-1 circumvented inhibition by IL-1 beta (0.1-1.0 U/ml). The interaction of cytokines and bone growth factors with osteoblasts are likely to be of critical importance in the regulation of bone mass at local inflammatory sites.