Antibodies to the IL-12 receptor beta 2 chain mark human Th1 but not Th2 cells in vitro and in vivo.

Great attention has been placed on the possibility of distinguishing Th1 from Th2 cells on the basis of differential expression of surface receptors. We have recently shown that the differential expression of the IL-12R beta 2 chain in Th1 and Th2 cells, as measured at the mRNA level, accounts for an important regulatory mechanism in the differentiation of the two cell subsets. In this study, we identify IL-12R expression at the protein level. We have generated an anti-IL-12R beta 2-specific mAb and analyzed IL-12R beta 2 expression on polarized Th cell populations generated in vitro and on T cells derived from patients with Th1- or Th2-mediated inflammatory conditions. Although IL-12R beta 2 was absent in freshly isolated PBMC and in cord blood cells, we were able to detect IL-12R beta 2 expression selectively in differentiated Th1 and T cytotoxic 1, but not Th2 or T cytotoxic 2 cells. In the presence of IL-12, cell surface expression of the IL-12R beta 2 subunit was readily detected on T cells after 24 h, reached the maximum at day 5, and declined thereafter. Most importantly, the anti-IL-12R beta 2 mAb recognizes lung T cells from patients with sarcoidosis, a disease characterized by a typical cell-mediated, Th1-type inflammatory response. In contrast, IL-12R beta 2 was absent in lung T cells from patients with allergic asthma, a disease characterized by a Th2-type inflammatory response. The mAb reported in this study should represent a powerful tool to investigate the role of Th1 and Th2 cells in inflammatory conditions and to monitor therapies aimed at altering the balance of Th cell subsets.

The role of Stat4 in species-specific regulation of Th cell development by type I IFNs.

Type I IFNs (IFN-alpha/beta), in addition to IL-12, have been shown to play an important role in the differentiation of human, but not mouse, Th cells. We show here that IFN-alpha/beta act directly on human T cells to drive Th1 development, bypassing the need for IL-12-induced signaling, whereas IFN-alpha cannot substitute IL-12 for mouse Th1 development. The molecular basis for this species specificity is that IFN-alpha/beta activate Stat4 in differentiating human, but not mouse, Th cells. Unlike IL-12, which acts only on Th1 cells, IFN-alpha/beta can activate Stat4 not only in human Th1, but also in Th2 cells. However, restimulation of human Th2 lines and clones in the presence of IFN-alpha does not induce the production of IFN-gamma. These results suggest that activation of Stat4, which is necessary for the differentiation of naive T cells into polarized Th1 cells, is not sufficient to induce phenotype reversal of human Th2 cells.

Effect of acetate, bicarbonate dialysis, and acetate-free biofiltration on nitric oxide synthesis: implications for dialysis hypotension.

The effect of acetate dialysis (AD), bicarbonate dialysis (BD), and acetate-free biofiltration (AFB) on nitric oxide (NO) synthesis and the implications for dialysis hypotension was studied. The finding that uremic plasma is a potent inducer of NO synthesis by endothelial cells in vitro suggested that the cardiovascular instability of dialysis patients might result from excessive NO formation. Cardiovascular instability is more frequent in patients undergoing AD than BD. To see whether these differences were attributable to NO, we studied the NO synthetic pathway ex vivo in patients undergoing different dialysis procedures. Five patients were treated, in a random order, with AD, BD, and AFB, a technique using a buffer-free dialysate and postdilution of a sterile bicarbonate solution. Each type of dialysis was used for 1 week, comprising three dialysis sessions. A polyacrylonitrile dialyzer was used for all three methods. Before and after the third dialysis, plasma was collected, added to [3H]L-arginine, and incubated with human umbilical vein endothelial cells (HUVECs) for 24 hours. NO synthesis was evaluated as [3H]L-citrulline formation. Plasma concentrations of interleukin-1beta (IL-1beta), a potent inducer of inducible NO synthase (iNOS) in endothelial cells, were also measured. Plasma collected from patients after AD stimulated endothelial NO synthesis more than plasma from the same patients before the dialysis session (pre-AD, 0.173+/-0.028 nmol/10(5) cells v post-AD, 0.280+/-0.093 nmol/10(5) cells; P < 0.05). A slight, although not significant, increase was also observed when HUVECs were incubated with plasma drawn after BD (pre-BD, 0.151+/-0.014 nmol/10(5) cells; post-BD, 0.230+/-0.055 nmol/10(5) cells). AFB did not aggravate the stimulatory effect of uremic plasma on endothelial NO synthesis (pre-AFB, 0.184+/-0.038 nmol/10(5) cells; post-AFB, 0.189+/-0.040 nmol/10(5) cells). Plasma IL-1beta was greater (P < 0.01) after AD than after BD and AFB (post-AD, 0.234+/-0.028 pg/mL; post-BD, 0.124+/-0.019 pg/mL; post-AFB, 0.120+/-0.013 pg/mL). With AD, there was a greater intradialytic decrease in systolic blood pressure than with BD or AFB. Weight and blood volume loss and sodium balance were similar in AD, BD, and AFB. These data were consistent with the possibility that NO and cytokines, released in excessive amounts during AD, may contribute to hemodynamic instability.

Analysis of the multiple interactions between IL-12 and the high affinity IL-12 receptor complex.

IL-12 is a heterodimeric cytokine, composed of a p40 and a p35 subunit, that exerts its biological effects by binding to specific cell surface receptors. Two IL-12R proteins, designated human IL-12 (huIL-12) receptor beta1 (huIL-12Rbeta1) and huIL-12Rbeta2, have been previously identified. These IL-12R individually bind huIL-12 with low affinity and in combination bind huIL-12 with high affinity and confer IL-12 responsiveness. In this study the interactions of hulL-12 with these two identified human IL-12R protein subunits are examined. The heterodimer-specific anti-huIL-12 mAb 20C2, which neutralizes huIL-12 bioactivity but does not block 125I-huIL-12 binding to huIL-12Rbeta1, blocked binding of huIL-12 to huIL-12Rbeta2. In contrast, anti-huIL-12Rbeta1 mAb 2B10 and mouse IL-12 p40 subunit homodimer (mo(p40)2) blocked 125I-huIL-12 binding to huIL-12Rbeta1, but not to huIL-12Rbeta2. Therefore, two classes of IL-12 inhibitors can be identified that differ in their ability to block huIL-12 interaction with either huIL-12Rbeta1 or huIL-12Rbeta2. Both mo(p40)2 and 20C2 blocked high affinity binding to huIL-12Rbeta1/beta2-cotransfected COS-7 cells, although, as previously reported, mo(p40)2 does not block high affinity binding to IL-12R on PHA-activated human lymphoblasts. Furthermore, these two classes of IL-12 inhibitors synergistically decreased hulL-12-stimulated proliferation and IFN-gamma production. Therefore, IL-12, in binding to the high affinity IL-12R, interacts with IL-12Rbeta1 primarily via regions on the IL-12 p40 subunit and with IL-12Rbeta2 via 20C2-reactive, heterodimer-specific regions of IL-12 to which the p35 and p40 subunits both contribute.

Clinical evaluation of a peritoneal dialysis solution with 33 mmol/L bicarbonate.

OBJECTIVE: To evaluate the efficacy and safety of a new peritoneal dialysis solution with 33 mmol/L bicarbonate. DESIGN: In an acute, prospective, randomized cross-over study, 8 patients were randomized in two groups of 4. On the first study day, the first group performed two consecutive 4-hour exchanges with a dialysis solution containing 35 mmol/L lactate: the first exchange with 13.6 g/L and the second with 38.6 g/L dextrose. On the second study day, the same type of exchanges were performed with bicarbonate. The second group underwent the same treatment, but used bicarbonate solutions on the first day and control solutions on the second study day. Thirty-three patients participated in a 2-month prospective and randomized study. After a 4-week baseline period using solutions containing 40 mmol/L lactate, the patients were dialyzed with either 33 mmol/L bicarbonate solutions or 40 mmol/L lactate solutions. SETTING: Peritoneal dialysis units at the University Hospital of Brescia and the Niguarda Hospital of Milan, Italy. RESULTS: Acute study: Control and bicarbonate solutions had similar effects on blood chemistries and peritoneal transport. Chronic study: Mean venous bicarbonate concentrations remained unchanged in the control group (26.6-27.2 mmol/L), but decreased significantly in the bicarbonate group from 28.8 mmol/L at the start of the study to 23.0 mmol/L after 2 months of bicarbonate administration. Other biochemical parameters remained unchanged. CONCLUSION: A peritoneal dialysis solution with a bicarbonate level of 33 mmol/L does not adequately correct uremic acidosis.

A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits.

We have identified a cDNA from a human phytohemagglutinin-activated lymphoblast library encoding a protein that binds 125I-labeled human interleukin 12 (125I-huIL-12) with a Kd of about 5 nM when expressed in COS-7 cells. When coexpressed in COS-7 cells with the previously identified IL-12 beta receptor (IL-12R beta) protein, two classes of 125I-huIL-12 binding sites were measured with Kds of about 55 pM and 8 nM, corresponding to the high- and low-affinity binding sites seen on phytohemagglutinin-activated lymphoblasts. This newly identified huIL-12R subunit is a member of the cytokine receptor superfamily, with closest resemblance to the beta-type cytokine receptor gp130 and the receptors for leukemia inhibitory factor and granulocyte colony-stimulating factor. Consequently, we have reclassified the previously identified IL-12R beta subunit as huIL-12R beta 1 and designated the newly identified subunit as huIL-12R beta 2. huIL-12R beta 2 is an 862-amino acid type I transmembrane protein with a 595-amino-acid-long extracellular domain and a cytoplasmic tail of 216 amino acids that contains three tyrosine residues. A cDNA encoding the mouse homolog of the huIL12R beta 2 protein has also been isolated. Human and mouse IL-12R beta 2 proteins show a 68% amino acid sequence identity. When expressed in COS-7 cells, huIL-12R beta 2 exists as a disulfide-linked oligomer with an apparent monomeric molecular weight of 130 kDa. Coexpression of the two identified IL-12R subunits in Ba/F3 cells conferred IL-12 responsiveness, and clones of these cotransfected Ba/F3 cells that grew continuously in the presence of IL-12 were isolated and designated LJM-1 cells. LJM-1 cells exhibited dose-dependent proliferation in response to huIL-12, with an ED50 of about 1 pM huIL-12. Interestingly, Ba/F3 cells transfected with IL-12R beta 2 alone proliferated in response to huIL-12 with an ED50 of about 50 pM, although a role for endogenous mouse IL-12R beta 1 in IL-12 signal transduction in these transfectants cannot be ruled out. These results demonstrate that the functional high-affinity IL-12R is composed of at least two beta-type cytokine receptor subunits, each independently exhibiting a low affinity for IL-12.

Evidence for multiple sites of interaction between IL-12 and its receptor.

We have previously described the identification of a protein, now designated IL-12R beta 1, that binds 125I-huIL-12 with a Kd of about 10 nM, corresponding to the low affinity 125I-huIL-12 binding sites seen on PHA-activated human lymphoblasts. Using expression cloning techniques, we have recently identified an additional IL-12-binding protein subunit, IL-12R beta 2, which binds 125I-huIL-12 with a Kd of about 5 nM when expressed alone in COS-7 cells. Coexpression of IL-12R beta 1 and IL-12R beta 2 in COS-7 cells results in formation of two classes of 125 I-huIL-12-binding sites with Kds of about 50 pM and 5 nM. Mouse IL-12 p40 subunit homodimer (mo(p40)2) blocked 125I-huIL-12 binding to human IL-12R beta 1, but did not inhibit binding to human IL-12R beta 2. In contrast, anti-huIL-12 monoclonal antibody 20C2, which does not block 125I-huIL-12 binding to human IL-12R beta 1, completely blocked binding to human IL-12R beta 2. These results demonstrate that two classes of IL-12 inhibitors, one that primarily blocks IL-12/IL-12R beta 1 interaction (e.g., mo(p40)2), and one that primarily blocks IL-12/IL-12R beta 2 interaction (e.g., 20C2), can be identified.

Randomized study with cyclosporine in kidney transplantation: 10-year follow-up.

This study presents the 10-yr follow-up results of a multicenter controlled trial on 108 recipients of cadaveric renal transplantation, randomized to receive cyclosporine (N = 55) or azathioprine (N = 53), both in combination with steroids. The 10-yr patient survival rate was 89% in the cyclosporine group and 83% in the azathioprine group (P = not significant [NS]); the 10-yr graft survival was 56% and 35%, respectively (log-rank test, P = 0.009). The half-life of grafts functioning after 1 yr was 15.4 +/- 3.9 versus 10.6 +/- 3.6, P = NS). The rate of early rejection in the cyclosporine group was significantly lower than that in the azathioprine group (0.30 versus 1.4, P < 0.01). Although the mean creatinine clearance rate was always higher in the azathioprine group, the decline in graft function from the first to the tenth yr was not significantly different between the two groups (-13.0 +/- 16.4 versus -12.3 +/- 19 mL/min, P = NS). In cadaveric renal transplantation, cyclosporine allows better graft survival than azathioprine, not only in the short term but also in the long term, with similar attrition of graft function for up to 10 yr.

Biological function and distribution of human interleukin-12 receptor beta chain.

We previously described the cloning of a cDNA encoding an interleukin-12 receptor (IL-12R) subunit, designated beta, that bound IL-12 with low affinity when expressed in COS cells. We now report that a pair of monoclonal antibodies (mAb), 2B10 and 2.4E6, directed against different epitopes on the IL-12R beta chain, when used in combination, strongly inhibited IL-12-induced proliferation of activated T cells, IL-12-induced secretion of interferon-gamma by resting peripheral blood mononuclear cells (PBMC), and IL-12-mediated lymphokine-activated killer cell activation. The mAb had no effect on lymphoblast proliferation induced by IL-2, -4, or -7. Thus, the IL-12R beta chain appears to be an essential component of the functional IL-12R on both T and natural killer (NK) cells. We previously observed that high affinity IL-12R were expressed on activated T and NK cells, but not B cells. Studies using flow cytometry and reverse transcription-polymerase chain reaction analysis showed that IL-12R beta chain was expressed on several human T, NK, and (surprisingly) B cell lines, but not on non-lymphohematopoietic cell lines. The Kit225/K6 (T cell) and SKW6.4 (B cell) lines were found to express the greatest amounts of IL-12R beta chain (800-2500 sites/cell); however, Kit225/K6 but not SKW6.4 cells bound IL-12. Similar to SKW6.4 B cells, activated tonsillar B lymphocytes expressed IL-12R beta chain but, consistent with previous results, did not display detectable IL-12 binding. Likewise, up to 72% of resting PBMC from normal volunteer donors expressed IL-12R beta, but did not bind measurable amounts of IL-12. These results indicate that expression of IL-12R beta is essential, but not sufficient, for expression of functional IL-12R. We speculate that expression of functional, high-affinity IL-12R may require the presence of a second subunit that is more restricted in its expression than IL-12R beta.

Long-term predictors of survival in essential mixed cryoglobulinemic glomerulonephritis.

We report the clinical outcome of 105 essential mixed cryoglobulinemia (EMC) patients with renal involvement collected throughout 25 years in three renal Units of Milan. The median follow-up was 72 months since renal biopsy and 131 months since the clinical onset of EMC. Patient survival was 49% at 10 years after renal biopsy. Forty-two patients died primarily from cardiovascular and liver disease or infection, whereas 15 patients developed chronic renal failure. Two patients had a complete remission of the disease while 15 had a remission only of renal signs. Thirty-one patients are alive with persistent renal and extrarenal manifestations. Anti-HCV antibodies were retrospectively detected in 34 patients and were present in 85% of them. This variable was not included in the statistical evaluation. At multivariate analysis, age older than 50 years, purpura, splenomegaly, cryocrit levels higher than 10%, C3 plasma levels lower than 54 mg/dl, and serum creatinine higher than 1.5 mg/dl were independent risk factors for death or dialysis. In conclusion, several factors may influence the outcome of patients with EMC nephritis. Markers of disease activity and an impaired renal function can herald a bad prognosis. It should be stressed, however, that only a minority of patients eventually develop renal failure, probably because in the most severe cases patients die earlier.

End-stage renal failure from renal amyloidosis of the AA type associated with giant lymph node hyperplasia (Castleman's disease).

A case of end-stage renal failure caused by renal amyloidosis of the AA type is reported. No chronic disease responsible for the deposition of reactive amyloid was detected until giant lymph node hyperplasia of the angiofollicular type was identified in a mediastinal mass. Amyloid was found within the tumour mass and was characterized by immunochemistry with monoclonal antibodies to be of the AA type. Castleman's disease should be added to the list of chronic diseases endangering renal function by inducing the production and tissue deposition of secondary (AA) amyloid.

Lymphocytic intracellular pH and Na+/H+ exchanger activity in hemodialysis patients.

We have evaluated intracellular pH (pHi) and Na+/H+ exchanger activity in peripheral lymphocytes from 16 patients on regular acetate hemodialysis. All the patients were taking oral NaHCO3 supplementation (30 mmol/day), to maintain predialysis arterial blood acid-base status within normal range (pH 7.36 +/- 0.02, PHCO3- 23.3 +/- 1.2 mM, pCO2 40.9 +/- 1.4 mm Hg). pHi was measured, using the fluorescent probe BCECF (2',7'-bis-carboxyethyl-5,6-carboxyfluorescein), both in nominal absence of bicarbonate (Hepes solution, pH 7.4; n = 10) and in the presence of HCO3-/CO2 buffer system (pH 7.4, [HCO3-] 25 mM, pCO2 40 mm Hg; n = 6). Predialysis pHi did not differ from controls when measured in the presence of HCO3-/CO2 (7.28 +/- 0.04 vs. 7.29 +/- 0.04, p = NS), but was lower in dialysis patients than in normal subjects (7.11 +/- 0.04 and 7.20 +/- 0.02, respectively; p < 0.05) when measured in Hepes solution. This suggested that bicarbonate-independent pHi regulation was abnormal in dialysis patients. To further characterize this abnormality of pHi regulation, lymphocytes were exposed to ethylisopropylamiloride, a specific Na+/H+ antiporter inhibitor, in Hepes solution; this maneuver induced a significantly lower decrement in pHi (0.04 +/- 0.04 vs. 0.15 +/- 0.03, p < 0.05) in dialysis patients than in controls, indicating reduced Na+/H+ exchanger activity in the patients. The rate of pHi recovery during the first 30 s after induction of various degrees of cell acidification (pHi range 6.2-7.0), which in the absence of HCO3-/CO2 is dependent on Na+/H+ exchanger activity, was also reduced in the patients as compared to controls (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression cloning of a human IL-12 receptor component. A new member of the cytokine receptor superfamily with strong homology to gp130.

A cDNA encoding a human IL-12R subunit was isolated by expression cloning. This subunit is a 662 amino acid type I transmembrane protein with an extracellular domain of 516 amino acids and a cytoplasmic domain of 91 amino acids. It is a member of the hemopoietin receptor superfamily and is most closely related over its entire length to gp130 and the receptors for granulocyte-CSF (G-CSF) and leukemia-inhibitory factor. When expressed in COS cells, this IL-12R subunit binds both human and murine IL-12 with an apparent affinity of 2 to 5 nM. The transfected COS cells express both monomers and disulfide-linked dimers or oligomers of the IL-12R subunit on their surface. However, unlike the IL-6-induced dimerization of gp130, the oligomerization of the IL-12R subunit is not dependent on binding of IL-12. Only the IL-12R subunit dimers/oligomers but not the monomers bind IL-12 with an affinity of 2 to 5 nM. A polyclonal antiserum raised against this receptor subunit specifically inhibits IL-12-induced proliferation of PHA-activated PBMC. The data are consistent with the hypotheses that 1) a dimer/oligomer of the cloned IL-12R subunit (IL-12R-beta) represents the low affinity IL-12 binding site identified on human lymphoblasts, 2) the cloned receptor subunit is involved in IL-12 signal transduction, and 3) an additional, as of yet unidentified subunit is required to generate a high affinity IL-12R complex.

Correction of hypokalemia with antialdosterone therapy in Gitelman's syndrome.

Six adult patients (4 females and 2 males, age range 26-57 years) with Gitelman's syndrome (GS) were treated with spironolactone 200-300 mg/day (n = 5) and/or amiloride 10-30 mg/day (n = 3) for 1-18 months. The patients had hypokalemia, hyperreninemia, chloride-resistant metabolic alkalosis, renal hypomagnesemia (n = 5), and hypocalciuria (n = 5). Free water clearance studies during maximal water diuresis and furosemide administration were suggestive of a solute reabsorptive defect beyond the loop of Henle. Antialdosterone therapy induced a significant increase of PK (from 2.6 +/- 0.4 to 3.4 +/- 0.4 mM; p < 0.0001) and a decrease of CK (from 21.4 +/- 13.2 to 10.6 +/- 4.8 ml/min, p < 0.02) and FEK (from 21.0 +/- 13.6 to 13.4 +/- 5.7%; p < 0.03); PMg increased from 1.38 +/- 0.38 to 1.64 +/- 0.21 mg/dl (p < 0.03) with a parallel fall of CMg (from 5.5 +/- 2.3 to 2.9 +/- 1.5 ml/min; p < 0.02) and FEMg (from 5.7 +/- 2.6 to 2.9 +/- 0.6%; p < 0.05); arterial blood pH and HCO3- did not change (P = plasma, C = clearance, FE = fractional excretion). The creatinine clearance fell (from 90.5 +/- 16.8 to 65.8 +/- 20.9 ml/min; p < 0.05), and Prenin rose (from 16.6 +/- 8.9 to 35.3 +/- 25.3 ng/ml/h; p < 0.02, as did Paldo (from 26.1 +/- 12.3 to 109 +/- 82.6 ng/dl; p < 0.01), indicating extracellular fluid volume contraction; however no significant clinical symptoms of hypovolemia ensued.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium metabolism and calciotropic hormone levels in Gitelman's syndrome.

In 10 adult patients (5 females and 5 males, aged 13-57 years) with Gitelman's syndrome (GS, or hypocalciuric variant of Bartter's syndrome, characterized by chronic renal hypokalemia, metabolic alkalosis, hypomagnesemia and hypocalciuria), parameters of Ca metabolism and calciotropic hormone levels were evaluated. Hypocalciuria was associated with a marked reduction of fractional excretion of ionized Ca (as compared with 16 sex- and age-matched controls) and normal filtered Ca load, as indicated by serum ionized Ca; hypocalciuria was thus the result of increased tubular reapsorption of filtered Ca. Plasma levels of total Ca were increased in GS (p < 0.02) but ionized Ca was not different from controls; percent fraction of ionized to total Ca was reduced, indicating increased Ca complexation and/or protein binding, possibly related to a metabolic alkalosis-induced increase of plasma albumin affinity for Ca. Levels of plasma total protein and albumin were similar in GS and controls. Despite similar ionized Ca levels, PTH1-84 was lower in GS than in controls, indicating abnormal ionized Ca-PTH relationship, possibly related to hypomagnesemia. Plasma 1,25(OH)2D levels were not different in GS and in controls, and intestinal fractional Ca absorption (evaluated with a simplified method using stable Sr as a tracer) was not reduced in 4 patients. However, in 5 patients bone mineral density in the forearm (3 patients) or lumbar spine (2 patient) was normal. Thus, despite chronic hypocalciuria and normal 1,25(OH)2D levels, Ca 'thesaurosis' does not occur in bones of GS patients; a likely explanation is that, despite normal 'fractional' intestinal Ca absorption, 'net' intestinal absorption is reduced, due to increased intestinal Ca secretion.

Production of monoclonal antibodies to prostromelysin (ProMMP-3) and establishment of a quantitative prostromelysin ELISA assay.

Human prostromelysin (59 kDa) was purified from the conditioned medium of IL-1-stimulated human dermal fibroblasts and anti-prostromelysin monoclonal antibodies were produced and identified by ELISA assay. Using prostromelysin, a C-terminally truncated recombinant form of prostromelysin consisting of amino acids 1-255, and their respective activated enzymes, we have begun mapping the epitopes recognized by these monoclonal antibodies. Various patterns of reactivity against the proenzymes and activated enzymes were observed. In further attempts to map the epitopes, we employed synthetic peptides representing hydrophilic regions of the primary amino acid sequence of prostromelysin. Our monoclonal antibodies did not recognize these peptides, suggesting that the antibodies may be recognizing conformational epitopes composed of non-linear portions of prostromelysin. Using these monoclonal antibodies, we have developed a quantitative prostromelysin sandwich ELISA assay.