Severe mycobacterial and Salmonella infections in interleukin-12 receptor-deficient patients

Interleukin-12 (IL-12) is a cytokine that promotes cell-mediated immunity to intracellular pathogens by inducing type 1 helper T cell (TH1) responses and interferon-gamma (IFN-gamma) production. IL-12 binds to high-affinity beta1/beta2 heterodimeric IL-12 receptor (IL-12R) complexes on T cell and natural killer cells. Three unrelated individuals with severe, idiopathic mycobacterial and Salmonella infections were found to lack IL-12Rbeta1 chain expression. Their cells were deficient in IL-12R signaling and IFN-gamma production, and their remaining T cell responses were independent of endogenous IL-12. IL-12Rbeta1 sequence analysis revealed genetic mutations that resulted in premature stop codons in the extracellular domain. The lack of IL-12Rbeta1 expression results in a human immunodeficiency and shows the essential role of IL-12 in resistance to infections due to intracellular bacteria.     

Interferon-gamma and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) are mutually antagonistic cytokines that stimulate CD4+ T cells to develop into either Th1 or Th2 cells. One feature of Th2 differentiation in mice is the loss of IL-12-induced Jak2 and Stat4 activation, which is accompanied by the inability to produce IFN-gamma in response to IL-12. In this report, we show that freshly isolated human T cells activated with phytohemagglutinin (PHA) in the presence of IL-4 exhibit a greatly diminished response to IL-12, whereas the IL-12 response of T cells activated with PHA plus IFN-gamma is enhanced. Radiolabeled IL-12 binding studies demonstrate that the impairment of T cell IL-12 responsiveness by IL-4 is associated with the down-regulation of high-affinity IL-12 receptor expression. In contrast, the enhancement of IL-12 responsiveness by IFN-gamma is associated with the upregulation of high-affinity IL-12 receptor expression. Through the use of a newly synthesized neutralizing antibody to the low-affinity IL-12 receptor beta subunit (IL-12Rbeta), we show that neither IL-4 nor IFN-gamma affect the expression of IL-12Rbeta, which we determine to be one of at least two low-affinity subunits required for high-affinity IL-12 binding. These findings suggest that IL-4 and IFN-gamma exert opposite effects on T cell IL-12 responsiveness by differentially modulating the expression of low-affinity IL-12 receptor subunits that are distinct from IL-12Rbeta and required, together with IL-12Rbeta, for high-affinity IL-12 binding and IL-12 responsiveness. This provides a basis for understanding the interplay between different cytokines at the level of cytokine receptor expression, and offers insight into one of the mechanisms governing Th1 and Th2 development.     

Selective suppression of interleukin-12 induction after macrophage receptor ligation

Interleukin (IL)-12 is a monocyte- and macrophage-derived cytokine that plays a crucial role in both the innate and the acquired immune response. In this study, we examined the effects that ligating specific macrophage receptors had on the induction of IL-12 by lipopolysaccharide (LPS). We report that ligation of the macrophage Fcgamma, complement, or scavenger receptors inhibited the induction of IL-12 by LPS. Both mRNA synthesis and protein secretion were diminished to near-undetectable levels following receptor ligation. Suppression was specific to IL-12 since IL-10 and tumor necrosis factor-alpha (TNF-alpha) production were not inhibited by ligating macrophage receptors. The results of several different experimental approaches suggest that IL-12 downregulation was due to extracellular calcium influxes that resulted from receptor ligation. First, preventing extracellular calcium influxes, by performing the assays in EGTA, abrogated FcgammaR-mediated IL-12(p40) mRNA suppression. Second, exposure of macrophages to the calcium ionophores, ionomycin or A23187, mimicked receptor ligation and inhibited IL-12(p40) mRNA induction by LPS. Finally, bone marrow-derived macrophages from FcR gamma chain-deficient mice, which fail to flux calcium after receptor ligation, failed to inhibit IL-12(p40) mRNA induction. These results indicate that the calcium influxes that occur as a result of receptor ligation are responsible for inhibiting the induction of IL-12 by LPS. Hence, the ligation of phagocytic receptors on macrophages can lead to a dramatic decrease in IL-12 induction. This downregulation may be a way of limiting proinflammatory responses of macrophages to extracellular pathogens, or suppressing the development of cell-mediated immunity to intracellular pathogens.     

Purification and characterization of the human interleukin-18 receptor

Interleukin (IL)-18 was identified as a molecule that induces IFN-gamma production and enhances NK cell cytotoxicity. In this paper, we report upon the purification and characterization of human IL-18 receptor (hIL-18R). We selected the Hodgkin's disease cell line, L428, as the most strongly hIL-18R-expressing cell line based on the results of binding assays. This binding was inhibited by IL-18 but not by IL-1beta. The dissociation constant (Kd) of 125I-IL-18 binding to L428 cells was about 18.5 nM, with 18,000 binding sites/cell. After immunizing mice with L428 cells and cloning, a single monoclonal antibody (mAb) against hIL-18R was obtained (mAb 117-10C). Sequentially, hIL-18R was purified from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-extracted L428 cells by wheat germ lectin-Sepharose 4B chromatography and mAb 117-10C-Sepharose chromatography. The internal amino acid sequences of hIL-18R all matched those of human IL-1 receptor-related protein (IL-1Rrp), the ligand of which was unknown to date. When expressed in COS-1 cells, the cDNA of IL-1Rrp conferred IL-18 binding properties on the cells and the capacity for signal transduction. From these results, we conclude that a functional IL-18 receptor component is IL-1Rrp.     

Pharmacokinetics and pharmacodynamics of recombinant human interleukin-12 in male rhesus monkeys

The pharmacokinetics and pharmacodynamics of recombinant human interleukin-12 (rHuIL-12) were investigated in male rhesus monkeys. The monkeys received a 40-min i.v. infusion of 42.5 micrograms/kg of recombinant human interleukin-12 on day 1 followed by a s.c. injection of the same dose on day 5. Serum samples were collected at appropriate time points and assayed for interleukin (IL-12) by an IL-12 capture bioassay, interferon (IFN-gamma) by an IFN-gamma enzyme-linked immunosorbent assay, and neopterin by a neopterin radioimmunoassay. After i.v. infusion, the systemic clearance rate of this protein was very slow (average, 3 ml/hr/kg). The volume of distribution at steady state ranged from 59 to 90 ml/kg. After the s.c. dose, the mean Cmax was 61 ng/ml and the mean Tmax was 18 hr. The absolute bioavailability was moderate (20-30%) after s.c. injection. By compartmental analysis, by using a two-compartment model the T 1/2 lambda 1 ranged from 0.2 to 5 hr and the T 1/2 lambda 2 ranged from 13 to 19 hr. When determining the percentage of the area of the serum concentration-time curve, per phase, > 85% of the protein was found in the lambda 2 phase. We selected IFN-gamma as one of the pharmacodynamic markers to study because it is produced by T-lymphocytes and natural killer cells in response to IL-12. In addition, once IFN-gamma is produced, it primes macrophages for tumor killing that in turn secrete neopterin. We show that within 24 to 48 hr after the i.v. dose, IFN-gamma concentrations are elevated in these monkeys (average, 300 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of interleukin-12, interleukin-15, and interleukin-2 on human immunodeficiency virus type 1 replication in vitro

Cytokines may have clinical utility as therapeutic agents for human immunodeficiency virus type 1 (HIV-1) infection and as an adjuvant for vaccines. The effect of interleukin-12 (IL-12) and IL-15 on in vitro HIV-1 replication was investigated. IL-12 and IL-15 at doses up to 10 ng/ml had little effect on basal HIV-1 p24 antigen production by chronically HIV-infected T (ACH-2) and monocytic (U1) cell lines. For ACH-2 cells stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml), IL-12 and IL-15 significantly increased p24 antigen production by 20 and 30%, respectively (n = 6). In contrast, IL-12 and IL-15 (10 ng/ml) treatment of PMA-stimulated U1 cells decreased p24 antigen production by 16 and 15%, respectively (n = 6). We next studied the effect of IL-12 and IL-15 on HIV-infected peripheral blood mononuclear cells (PBMCs). In 10 HIV-seropositive patients' PBMCs cocultured with mitogen-activated HIV-seronegative donor cells, two patterns of p24 antigen production were observed in response to IL-2: low (p24 antigen production < 10(3) pg/ml; n = 8) and high (p24 antigen production > 10(3) pg/ml; n = 2) response. For the low-response pattern, IL-12 and IL-15 increased viral replication by 97-fold and 100-fold, respectively (P = 0.05 and 0.004, respectively). For the high-response pattern, both IL-12 and IL-15 suppressed HIV replication. The effect of IL-2, IL-12, and IL-15 on acute in vitro infection by HIV-1JRCSF was also examined. IL-12 did not increase p24 antigen production above basal levels while IL-2 and IL-15 significantly enhanced p24 antigen production (by approximately 2-fold). In conclusion, IL-12 and IL-15 may have differential effects on latent and acute HIV infection, and their ability to enhance HIV production may depend on cell activation. Thus, the use of these cytokines may be dictated by the clinical state of the patient.     

Increased interleukin-12 levels in human cerebrospinal fluid following severe head trauma

Light (LM) and electron microscopy (EM) were used to investigate the structural relationship between enteric nerves and the population of immune cells in the mouse small bowel. By LM, the osmium-zinc iodide procedure was used for visualizing nerve fibers; the incidence of nerve-plasma cell contacts in the mucosa and submucosa was calculated to be approximately 4 times and, respectively, 3 times greater than would be expected by chance alone (P < 0.0001). EM revealed close, synaptic-like contacts between axonal varicosities and plasma cells or B immunoblasts. The results presented here provide systematic quantitative evidence that a structural foundation for communication between nerve fibers and B cells exists in the mouse small bowel.     

Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist

OBJECTIVE: To evaluate the efficacy and safety of interleukin-1 receptor antagonist (IL-1Ra) in patients with rheumatoid arthritis (RA). METHODS: Patients with active and severe RA (disease duration <8 years) were recruited into a 24-week, double-blind, randomized, placebo-controlled, multicenter study. Doses of nonsteroidal antiinflammatory drugs and/or oral corticosteroids (< or =10 mg prednisolone daily) remained constant throughout the study. Any disease-modifying antirheumatic drugs that were being administered were discontinued at least 6 weeks prior to enrollment. Patients were randomized to 1 of 4 treatment groups: placebo or a single, self-administered subcutaneous injection of IL-1Ra at a daily dose of 30 mg, 75 mg, or 150 mg. RESULTS: A total of 472 patients were recruited. At enrollment, the mean age, sex ratio, disease duration, and percentage of patients with rheumatoid factor and erosions were similar in the 4 treatment groups. The clinical parameters of disease activity were similar in each treatment group and were consistent with active and severe RA. At 24 weeks, of the patients who received 150 mg/day IL-1Ra, 43% met the American College of Rheumatology criteria for response (the primary efficacy measure), 44% met the Paulus criteria, and statistically significant improvements were seen in the number of swollen joints, number of tender joints, investigator's assessment of disease activity, patient's assessment of disease activity, pain score on a visual analog scale, duration of morning stiffness, Health Assessment Questionnaire score, C-reactive protein level, and erythrocyte sedimentation rate. In addition, the rate of radiologic progression in the patients receiving IL-1Ra was significantly less than in the placebo group at 24 weeks, as evidenced by the Larsen score and the erosive joint count. IL-1Ra was well tolerated and no serious adverse events were observed. An injection-site reaction was the most frequently observed adverse event, and this resulted in a 5% rate of withdrawal from the study among those receiving IL-1Ra at 150 mg/day. CONCLUSION: This study confirmed both the efficacy and the safety of IL-1Ra in a large cohort of patients with active and severe RA. IL-1Ra is the first biologic agent to demonstrate a beneficial effect on the rate of joint erosion.     

Regulation of interleukin-13 receptor constituents on mature human B lymphocytes

Human B cells stimulated through both their immunoglobulin and CD40 receptors up-regulate 745 +/- 51 interleukin (IL)-13 ligand binding sites with an affinity of 0.91 +/- 0.08 nM within 24 h. IL-13 binds primarily to the IL-13Ralpha1 with subsequent sequestration of the IL-4Ralpha into the complex. IL-13Ralpha1 may also be found in those receptors capable of binding IL-4. gamma chain (gammac) participates in receptors capable of binding IL-4 but is not found in association with bound IL-13. Dimeric receptors composed of the IL-4Ralpha complexed with either the IL-13Ralpha1 or gammac occur simultaneously within defined B cell populations. mRNAs for all receptor constituents are increased subsequent to immunoglobulin stimulation alone, while maximal expression of IL-13Ralpha1 is more dependent upon co-stimulation of immunoglobulin and CD40 receptors. mRNA levels for IL-13Ralpha1 vary over a wider range subsequent to surface stimulation than other receptor components. Although gammac is not bound to IL-13 in B cells under the conditions evaluated, it may influence IL-13 binding by competing with IL-13Ralpha1 for association/sequestration with the IL-4Ralpha chain. IL-13Ralpha2 does not participate in the IL-13 receptor that is up-regulated upon activation of quiescent tonsillar B lymphocytes, although mRNA for the protein may be found in the centroblastic fraction of tonsillar cells.     

Immune complexes are potent inhibitors of interleukin-12 secretion by human monocytes

We have studied the effect of immune complexes (IC) on interleukin (IL)-12 secretion by human monocytes in vitro. Two experimental models of IC were used. IC formed of tetanus toxoid and polyclonal anti-tetanus toxoid antiserum as well as heat-aggregated human serum IgG almost completely inhibited IL-12 (p70 and p40) secretion induced by interferon-gamma and lipopolysaccharide in human blood-derived monocytes. Neutralizing anti-IL-10 antibodies plus indomethacin restored IL-12 secretion in the presence of IC to a high extent, indicating that IL-10 and prostaglandin (PG) partially mediate the IC-induced inhibition of IL-12 secretion. However, neutralization of tumor necrosis factor (TNF)-alpha by specific antibodies also incompletely restored IL-12 secretion. Indeed, monocytes secrete high levels of TNF-alpha upon stimulation by IC. We found that exogenously added TNF-alpha caused a profound inhibition of monocytic IL-12 secretion in the absence of IC, again mediated via the induction of IL-10 and PG. In summary, IC inhibit IL-12 secretion via TNF-alpha-induced IL-10 and PG synthesis. We conclude that IC, typically appearing in the course of chronic inflammatory processes, may influence the balance between Th1 and Th2 responses and may thus contribute to a deprivation of cell-mediated immune responses.     

Elevated interleukin-10-to-interleukin-12 ratio in feline immunodeficiency virus-infected cats predicts loss of type 1 immunity to Toxoplasma gondii

Similar to human immunodeficiency virus, feline immunodeficiency virus (FIV) induces immunodeficiency and enhanced susceptibility to secondary pathogens. To explore cytokine alterations in lentivirus immunodeficiency, constitutive mRNA expression was measured in lymph nodes of healthy and FIV-infected cats before and after challenge with Toxoplasma gondii. Cytokine mRNA expression was similar in control and FIV-infected cats during the first 10 weeks after infection. At 16 weeks, interferon (IFN)-gamma, tumor necrosis factor-alpha, and interleukin (IL)-10 mRNA were increased in FIV-infected cats. Challenge with T. gondii induced an increase in IL-2, IFN-gamma, and IL-12 in the lymph nodes of control cats, whereas IFN-gamma and IL-10 but not IL-2 or IL-12 increased in the lymph nodes of FIV-T. gondii coinfected cats. These results indicate that FIV immunodeficiency may derive from a failure to generate an IL-12-dependent type 1 response and that an elevated level of IL-10 mRNA expression is a predictor of lentivirus immunodeficiency.     

Differential regulation of the interleukin-12 receptor during the innate immune response to Leishmania major

Previous studies have shown the central role of interleukin 12 (IL-12) in the development of resistance to Leishmania major infection in C3H mice. We now show that during the innate immune response the lymph node cells of L. major-infected C3H mice upregulate the IL-12 receptor on CD4(+), CD8(+), and B220(+) cells. An increase in the ability of the lymph node cells to bind IL-12 correlates with 9.3- and 4.6-fold increases in the mRNA expression levels of the IL-12Rbeta1 and -beta2 subunits, respectively. In contrast, BALB/c mice, which are susceptible to L. major infection, have no increase in the ability of the lymph node cells to bind IL-12 and correspondingly smaller increases in the mRNA expression levels of the IL-12Rbeta1 and -beta2 subunits of 2- and 1.5-fold, respectively. Neutralizing IL-4 and the administration of exogenous IL-12 upregulate IL-12R expression in BALB/c mice, while the neutralization of IL-12 in C3H mice blocks increased IL-12 receptor expression. These experiments reveal an important role for the regulation of the IL-12 receptor during the innate immune response after infection of mice with a pathogen.     

Phase I trial of subcutaneous recombinant human interleukin-12 in patients with advanced renal cell carcinoma

Patients with advanced renal cell carcinoma were treated in a Phase I trial with escalating doses of recombinant human interleukin-12 (rHuIL-12) given on days 1, 8, and 15 of each 28-day cycle. Treatment in the initial dose scheme consisted of a fixed dose with dose levels of 0.1, 0.5, and 1.0 microg/kg given to cohorts composed of three or six patients. On the basis of the toxicity profile, a second scheme (up-titration) was undertaken wherein rHuIL-12 was escalated for each patient from week 1 to week 2, to a target dose given week 3 and thereafter; cohort target dose levels were 0.5, 0.75, 1.0, 1.25, and 1.5 microg/kg. Fifty-one patients were treated: 32 (63%) had prior cytokine therapy and 19 (37%) had received no prior systemic therapy. The maximum tolerated dose for the fixed dose scheme was 1.0 microg/kg. Dose-limiting toxicities included increase in transaminase concentration, pulmonary toxicity, and leukopenia. The most severe toxicities occurred with the first injection and were milder upon further treatment. With the up-titration dose scheme, the maximum tolerated dose was reached at 1.5 microg/kg, and dose-limiting toxicity consisted of an increase in serum transaminase levels. At the maximum tolerated dose of 1.5 microg/kg, serum IL-12 levels increased to a mean peak level of 706 pg/ml. Serum levels of IFN-gamma increased to a mean peak level of about 200 pg/ml at 24 h after the first maintenance dose of 1.5 microg/kg. The best responses were as follows: one patient had complete response, 34 patients were stable, 14 patients showed progression, and 1 patient was inevaluable. In conclusion, rHuIL-12 was relatively well tolerated when administered by s.c. injection. The recommended dose according to the up-titration schedule of rHuIL-12 (microg/kg) for Phase II trials was as follows: cycle 1, 0.1 (day 1), 0.5 (day 8), 1.25 (day 15); cycle 2 onwards, 1.25. Phase II trials of rHuIL-12 were initiated in previously untreated patients with renal cell carcinoma and in patients with melanoma.     

Induction of immunity against experimental tuberculosis with mycobacterial mannophosphoinositides encapsulated in liposomes containing lipid A

Auditory brainstem responses (ABR) to high-frequency (> or = 8 kHz) tone-burst stimuli have shown potential for objective early detection of ototoxicity. In the case of ill, unresponsive, or otherwise difficult-to-test individuals, the patient group for whom this test is targeted, a threshold-seeking process can be too lengthy. A new method is described for obtaining responses to several high-frequency tone bursts in the same amount of time as that used in obtaining a single responses. Using 10 normal-hearing subjects, four high-frequency tone-burst stimuli (14, 12, 10, and 8 kHz) were presented singly, then in a multiple-stimulus sequence with onsets separated by 10 msec. Wave V response latencies from the multiple-stimulus sequences are compared to those presented singly, with small but statistically significant longer latencies observed for all stimuli following the initial stimulus (14 kHz) in the multiple sequence. Test-retest reliability was comparable between multiple and single conditions. These findings support the development of this technique for clinical auditory monitoring.     

Innate immunity to Toxoplasma gondii is influenced by gender and is associated with differences in interleukin-12 and gamma interferon production

Given that differences between the sexes in relative susceptibility to parasitic infections have been noted, this study further elucidates the mechanisms responsible by demonstrating that male SCID mice are more resistant than female mice to infection with Toxoplasma gondii and that this difference correlates with enhanced innate immune responses in these animals. Male SCID mice exhibited longer survival times, lower parasite burdens, and less severe pathological changes postinfection. An immunological basis for these differences is demonstrated in that these animals produced interleukin-12 more rapidly and exhibited higher levels of gamma interferon earlier postinfection.     

Interleukin-1 beta enhances interleukin-1 receptor antagonist content in human somatotroph adenoma cell cultures

In addition to the well-known modulation of immune and inflammatory responses, the interleukin-1 (IL-1) system has been shown to be involved in the regulation of anterior pituitary hormone secretion and growth. We previously demonstrated that IL-1 receptor antagonist (IL-1ra) is expressed in human pituitary adenomas cultured in vitro. In the present study, we investigated the regulation of IL-1ra protein by IL-1 beta (1-100 U/mL) in human somatotroph adenomas (n = 9) cultured for 12-48 h. IL-1 beta significantly enhanced the concentration of IL-1ra dose dependently in the somatotroph adenoma cell lysates, whereas IL-1ra concentrations remained unchanged in the culture supernatants. Furthermore, basal IL-1ra concentrations were significantly higher in the cell lysates compared with the corresponding culture supernatants. The regulation of IL-1ra in somatotroph adenoma cells is different from human cultured monocytes, in which IL-1 beta significantly stimulated IL-1ra secretion into the culture supernatants, and no change of intracellular IL-1ra content was observed. Incubation of the somatotroph adenoma cells with 100 U/mL IL-1 beta did not result in a change of GH concentrations in the culture supernatants. Enhancement of intracellular IL-1ra protein by IL-1 beta may represent a mechanism intrinsic to somatotroph adenoma cells to counterregulate the response to IL-1 beta on hormone secretion or cellular growth.