We used a sensitive method based on tetramers of peptide and major histocompatibility complex II (pMHCII) to determine whether CD4(+) memory T cells resemble the T helper type 1 (T(H)1) and <em>interleukin</em> 17 (IL-17)-producing T helper (T(H)17) subsets described in vitro. Intravenous or intranasal infection with Listeria monocytogenes induced pMHCII-specific CD4(+) naive T cells to proliferate and produce effector cells, about 10% of which resembled T(H)1 or T(H)17 cells, respectively. T(H)1 cells were also present among the memory cells that survived 3 months after infection, whereas T(H)17 cells disappeared. The short lifespan of T(H)17 cells was associated with small amounts of the antiapoptotic protein Bcl-2, the IL-<em>15</em> receptor and the receptor CD27, and little homeostatic proliferation. These results suggest that T(H)1 cells induced by intravenous infection are more efficient at entering the memory pool than are T(H)17 cells induced by intranasal infection.

Cellular homeostasis requires a balance between cell production, cell survival, and cell death. Production of natural killer (NK) cells from bone marrow precursor cells requires <em>interleukin</em> <em>15</em> (IL-<em>15</em>); however, very little is known about the factors controlling survival of mature NK cells in vivo. Because mice deficient in IL-<em>15</em> (IL-<em>15</em>(-/-) mice) fail to develop NK cells, it is not known whether mature NK cells can survive in an environment lacking IL-<em>15</em>. We hypothesized that IL-<em>15</em> might indeed be required for survival of mature NK cells in vivo. Freshly isolated NK cells labeled with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) were adoptively transferred into IL-<em>15</em>(-/-) mice and littermate control (IL-<em>15</em>(+/-)) mice. Within 36 hours after transfer, NK cells were detected in both IL-<em>15</em>(-/-) and IL-<em>15</em>(+/-) mice; however, significantly more (P <.003) CFSE-positive (CFSE(+)) NK cells were found in control mice than in IL-<em>15</em>(-/-) mice. By 5 days, similar numbers of CFSE(+) NK cells were still easily detected in IL-<em>15</em>(+/-) mice, whereas no CFSE(+) NK cells survived in IL-<em>15</em>(-/-) mice. Furthermore, mice with severe combined immunodeficiency treated with the Fab fragment of a blocking antibody recognizing a signaling subunit of the IL-<em>15</em> receptor, IL-2/<em>15</em>Rbeta, had a significant ( approximately 90%) loss of NK cells compared with control mice. Finally, NK cells from Bcl-2 transgenic mice that were adoptively transferred into IL-<em>15</em>(-/-) mice did survive. These results show conclusively that IL-<em>15</em> is required for mature NK cell survival in vivo and suggest that IL-<em>15</em> mediates its effect on NK cell survival by means of Bcl-2.

OBJECTIVE

Abnormal cytokine expression accompanies myelofibrosis and might be a therapeutic target for Janus-associated kinase (JAK) inhibitor drugs. This study describes the spectrum of plasma cytokine abnormalities in primary myelofibrosis (PMF) and examines their phenotypic correlates and prognostic significance.

METHODS

Patients included in this study were required to have archived plasma, bone marrow biopsy, and cytogenetic information available at the time of first referral to the Mayo Clinic. Multiplex biometric sandwich immunoassay was used to measure plasma levels of 30 cytokines.

RESULTS

In total, 127 PMF patients were studied; comparison with normal controls (n = 35) revealed significantly increased <em>interleukin</em>-1β (IL-1β), IL-1RA, IL-2R, IL-6, IL-8, IL-10, IL-12, IL-13, IL-<em>15</em>, tumor necrosis factor α (TNF-α), granulocyte colony-stimulating factor (G-CSF), interferon alfa (IFN-α), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, hepatocyte growth factor (HGF), IFN-γ-inducible protein 10 (IP-10), monokine induced by IFN-γ (MIG), monocyte chemotactic protein 1 (MCP-1), and vascular endothelial growth factor (VEGF) levels and decreased IFN-γ levels. In treatment-naive patients (n = 90), increased levels of IL-8 (P < .001), IL-2R (P < .001), IL-12 (P < .001), IL-<em>15</em> (P = .001), and IP-10 (P = .003) were independently predictive of inferior survival. A similar multivariable analysis that included all 127 study patients confirmed the prognostic value of these five cytokines, and IL-8, IL-2R, IL-12, and IL-<em>15</em> remained significant when risk stratification, according to the recently revised Dynamic International Prognostic Scoring System (DIPSS plus), was added to the multivariable model. Leukemia-free survival was predicted by IL-8, which was also the only cytokine associated with ≥ 1% circulating blasts. Other cytokine-phenotype associations included increased IL-8 and constitutional symptoms; IL-2R, IL-12, and transfusion need; IL-2R, IL-8, and leukocytosis; IP-10 and thrombocytopenia; HGF, MIG, IL-1RA, and marked splenomegaly; and IL-1RA, IL-2R, IP-10, MIP-1β, and JAK2V617F. A two-cytokine (IL-8/IL-2R) -based risk categorization delineated prognostically different groups within specific DIPSS plus risk categories.

CONCLUSIONS

This study signifies the presence of specific cytokine-phenotype associations in PMF and a prognostically relevant plasma cytokine signature that might prove useful as a laboratory tool for predicting and monitoring treatment response.

Biochemical studies of signaling mediated by many cytokine and growth factor receptors have implicated members of the Jak family of tyrosine kinases in these pathways. Specifically, Jak3 has been shown to be associated with the <em>interleukin</em>-2 (IL-2) receptor gamma chain, a component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-<em>15</em>. Mice lacking Jak3 showed a severe block in B cell development at the pre-B stage in the bone marrow. In contrast, although the thymuses of these mice were small, T cell maturation progressed relatively normally. In response to mitogenic signals, peripheral T cells in Jak3-deficient mice did not proliferate and secreted small amounts of IL-2. These data demonstrate that Jak3 is critical for the progression of B cell development in the bone marrow and for the functional competence of mature T cells.

OBJECTIVE

We studied the effect of the Mediterranean diet on plasma levels of C-reactive protein (CRP), white blood cell counts, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, amyloid A, fibrinogen, and homocysteine.

BACKGROUND

To the best of our knowledge, the mechanism(s) by which the Mediterranean diet reduces cardiovascular risk are not well understood.

METHODS

During the 2001 to 2002 period, we randomly enrolled 1,514 men (18 to 87 years old) and 1,528 women (18 to 89 years old) from the Attica area of Greece (of these, 5% of men and 3% of women were excluded because of a history of cardiovascular disease). Among several factors, adherence to the Mediterranean diet was assessed by a diet score that incorporated the inherent characteristics of this diet. Higher values of the score meant closer adherence to the Mediterranean diet.

RESULTS

Participants who were in the highest tertile of the diet score had, on average, 20% lower CRP levels (p = 0.015), 17% lower IL-6 levels (p = 0.025), 15% lower homocysteine levels (p = 0.031), 14% lower white blood cell counts (p = 0.001), and 6% lower fibrinogen levels (p = 0.025), as compared with those in the lowest tertile. The findings remained significant even after various adjustments were made. Borderline associations were found regarding TNF-alpha (p = 0.076), amyloid A levels (p = 0.19), and diet score.

CONCLUSIONS

Adherence to the traditional Mediterranean diet was associated with a reduction in the concentrations of inflammation and coagulation markers. This may partly explain the beneficial actions of this diet on the cardiovascular system.

BACKGROUND

Patients with non-alcoholic steatohepatitis (NASH) have increased mortality and liver-related complications. In contrast, simple steatosis is considered benign and non-progressive.

OBJECTIVE

To investigate disease progression in patients with different degrees of non-alcoholic fatty liver disease (NAFLD) activity.

METHODS

Prospective longitudinal hospital-based cohort study.

METHODS

Fifty-two patients (age 44+/-9 years) with biopsy-proven NAFLD had liver biopsies repeated at month 36.

RESULTS

Among 13 patients with simple steatosis at baseline, 2 (<em>15</em>%) had a normal liver at month 36, 3 (23%) continued to have simple steatosis, 5 (39%) developed borderline NASH and 3 (23%) developed NASH. Among 22 patients with borderline NASH at baseline, 4 (18%) had simple steatosis and 13 (59%) had borderline NASH at month 36, while 5 (23%) developed NASH. Among 17 patients with NASH at baseline, 10 (59%) continued to have NASH and 6 (35%) had borderline NASH at month 36. Only 1 (6%) patient regressed to simple steatosis. Overall, 14 (27%) patients had fibrosis progression, 25 (48%) had static disease, and 13 (25%) had fibrosis regression. Reduction in body mass index and waist circumference was independently associated with non-progressive disease activity and fibrosis. The baseline serum levels and month 36 changes in adiponectin, tumour necrosis factor alpha, <em>interleukin</em> 6 and leptin were not associated with disease progression. Serum cytokeratin-18 fragment level reflected disease activity and its change correlated with the change in NAFLD activity score (R=0.51, p<0.001).

CONCLUSIONS

Patients with simple steatosis may still develop NASH and fibrosis progression. Weight reduction is associated with non-progressive disease. All patients with NAFLD should undergo periodic assessment and lifestyle modification.

BACKGROUND

Cytotoxic T lymphocyte-associated antigen (CTLA)-4 can inhibit T-cell responses and is involved in tolerance against self antigens. We previously reported autoimmune manifestations and objective cancer regressions in patients with metastatic melanoma treated with CTLA-4 blockade. The possibility of activating tumor-reactive T cells while removing inhibitory activity with CTLA-4 blockade has stimulated interest in using anti-CTLA-4 antibodies in combination with other cancer immunotherapies to improve clinical outcomes. In this study, we assessed the antitumor activity and autoimmune toxicity of CTLA-4 blockade in combination with an immune-activating stimulus, interleukin (IL)-2, in patients with metastatic melanoma.

METHODS

Thirty-six patients received anti-CTLA-4 antibody every 3 weeks. Three patients per cohort received doses of .1, .3, 1.0, and 2.0 mg/kg. Twenty-four patients received 3.0 mg/kg. All patients received IL-2 therapy (720,000 IU/kg every 8 hours to a maximum of 15 doses).

RESULTS

Eight patients (22%) experienced objective tumor responses (three complete and five partial), including metastases in the lungs, lymph nodes, mediastinum, and subcutaneous tissues. Six of the eight patients have ongoing objective responses at 11 to 19 months. Five patients (14%) developed grade III/IV autoimmune toxicities secondary to anti-CTLA-4 administration, including four patients with enterocolitis and one with arthritis and uveitis.

CONCLUSIONS

There is not evidence to support a synergistic effect of CTLA-4 blockade plus IL-2 administration, because the 22% objective response rate is that expected from the sum of these two agents administered alone. Durable cancer regressions were seen in patients treated with this combination.

OBJECTIVE

This article reviews recent developments in cytokine biology that are relevant to clinical psychiatry.

METHODS

The authors reviewed English-language literature of the last <em>15</em> years that pertains to the biology of cytokines with emphasis on central nervous system effects in general and psychiatric disorders in particular.

RESULTS

Growing evidence suggests that, in addition to providing communication between immune cells, specific cytokines play a role in signaling the brain to produce neurochemical, neuroendocrine, neuroimmune, and behavioral changes. This signaling may be part of a generalized, comprehensive mechanism to mobilize resources in the face of physical and/or psychological stress and to maintain homeostasis. The clinical implications of these findings are far-reaching and include a possible role for cytokines in the pathophysiology of specific psychiatric disorders such as major depression, schizophrenia, and Alzheimer's disease. The effects of cytokines in the central nervous system may provide a possible mechanism for the "sickness behavior" of patients with severe infection or cancer, as well as for the neuropsychiatric adverse effects of treatment with interferons and interleukins.

CONCLUSIONS

A better understanding of the role of cytokines in various brain activities will enhance knowledge of specific psychobiological mechanisms in health and disease and provide opportunities for novel treatment interventions.

Type I interferons (IFNs) are cytokines exhibiting antiviral and antitumor effects, including multiple activities on immune cells. However, the importance of these cytokines in the early events leading to the generation of an immune response is still unclear. Here, we have investigated the effects of type I IFNs on freshly isolated granulocyte/macrophage colony-stimulating factor (GM-CSF)-treated human monocytes in terms of dendritic cell (DC) differentiation and activity in vitro and in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) mice. Type I IFNs induced a surprisingly rapid maturation of monocytes into short-lived tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-expressing DCs endowed with potent functional activities, superior with respect to the <em>interleukin</em> (IL)-4/GM-CSF treatment, as shown by FACS((R)) analyses, mixed leukocyte reaction assays with allogeneic PBLs, and lymphocyte proliferation responses to HIV-1-pulsed autologous DCs. Type I IFN induced IL-<em>15</em> production and strongly promoted a T helper cell type 1 response. Notably, injection of IFN-treated HIV-1-pulsed DCs in SCID mice reconstituted with autologous PBLs resulted in the generation of a potent primary immune response, as evaluated by the detection of human antibodies to various HIV-1 antigens. These results provide a rationale for using type I IFNs as vaccine adjuvants and support the concept that a natural alliance between these cytokines and monocytes/DCs represents an important early mechanism for connecting innate and adaptive immunity.

12/<em>15</em>-Lipoxygenases (12/<em>15</em>-LOX) are members of the LOX family, which are expressed in mammals by monocytes and macrophages following induction by the T helper type 2 cytokines, <em>interleukins</em>-4 and -13. They oxygenate free polyenoic fatty acids but also ester lipids and even complex lipid-protein assemblies such as biomembranes and lipoproteins. The primary oxidation products are either reduced by glutathione peroxidases to corresponding hydroxy derivatives or metabolized into secondary oxidized lipids including leukotrienes, lipoxins and hepoxilins, which act as lipid mediators. Examination of knockout and transgenic animals revealed important roles for 12/<em>15</em>-LOX in inflammatory diseases, including atherosclerosis, cancer, osteoporosis, angiotension II-dependent hypertension and diabetes. In vitro studies suggested 12/<em>15</em>-LOX products as coactivators of peroxisomal proliferator activating-receptors (PPAR), regulators of cytokine generation, and modulators of gene expression related to inflammation resolution. Despite much work in this area, the biochemical mechanisms by which 12/<em>15</em>-LOX regulates physiological and pathological immune cell function are not fully understood. This review will summarize the biochemistry and tissue expression of 12/<em>15</em>-LOX and will describe the current knowledge regarding its immunobiology and regulation of inflammation.

<em>Interleukins</em> (IL) -1 beta and -1 alpha and tumor necrosis factor (TNF-alpha) were measured by radioimmunoassay in plasma samples from 44 healthy individuals, <em>15</em> patients in septic shock, and 6 volunteers infused with endotoxin. Plasma IL-1 alpha levels were low (40 pg/ml) or undetectable in all situations. In 67% of the healthy subjects, plasma IL-1 beta levels were less than 70 pg/ml. Septic patients had higher plasma IL-1 beta levels (120 +/- 17 pg/ml, P = .001); those of surviving patients were higher than those of patients who died (P = .05). Plasma TNF-alpha concentrations in septic individuals were elevated (119 +/- 30 pg/ml) and correlated with severity of illness (r = .73, P = .003), but no correlation was observed between plasma IL-1 beta and TNF-alpha concentrations in individual samples. Infusion of endotoxin caused a twofold elevation of IL-1 beta, from a baseline of 35 +/- 5 pg/ml to a maximum of 69 +/- 27 pg/ml at 180 min (P less than .05). Peak TNF-alpha levels after endotoxin infusion were <em>15</em> times higher than IL-1 beta levels, were attained more rapidly (90 min), and as with the septic patients, did not correlate with IL-1 beta levels. These data support the concept that plasma IL-1 beta and TNF-alpha concentrations are regulated independently and are associated with different clinical outcomes.

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of <em>interleukin</em>-7 (IL-7) and IL-<em>15</em>. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

OBJECTIVE

<em>Interleukin</em>-<em>15</em> (IL-<em>15</em>) has significant potential in cancer immunotherapy as an activator of antitumor CD8 T and natural killer (NK) cells. The primary objectives of this trial were to determine safety, adverse event profile, dose-limiting toxicity, and maximum-tolerated dose of recombinant human IL-<em>15</em> (rhIL-<em>15</em>) administered as a daily intravenous bolus infusion for 12 consecutive days in patients with metastatic malignancy.

METHODS

We performed a first in-human trial of Escherichia coli-produced rhIL-<em>15</em>. Bolus infusions of 3.0, 1.0, and 0.3 μg/kg per day of IL-<em>15</em> were administered for 12 consecutive days to patients with metastatic malignant melanoma or metastatic renal cell cancer.

RESULTS

Flow cytometry of peripheral blood lymphocytes revealed dramatic efflux of NK and memory CD8 T cells from the circulating blood within minutes of IL-<em>15</em> administration, followed by influx and hyperproliferation yielding 10-fold expansions of NK cells that ultimately returned to baseline. Up to 50-fold increases of serum levels of multiple inflammatory cytokines were observed. Dose-limiting toxicities observed in patients receiving 3.0 and 1.0 μg/kg per day were grade 3 hypotension, thrombocytopenia, and elevations of ALT and AST, resulting in 0.3 μg/kg per day being determined the maximum-tolerated dose. Indications of activity included clearance of lung lesions in two patients.

CONCLUSIONS

IL-<em>15</em> could be safely administered to patients with metastatic malignancy. IL-<em>15</em> administration markedly altered homeostasis of lymphocyte subsets in blood, with NK cells and γδ cells most dramatically affected, followed by CD8 memory T cells. To reduce toxicity and increase efficacy, alternative dosing strategies have been initiated, including continuous intravenous infusions and subcutaneous IL-<em>15</em> administration.

Graft-versus-host disease (GVHD) is caused by alloreactive donor T cells that trigger host tissue injury. GVHD develops over weeks or months, but how this immune response is maintained over time is unknown. In mouse models of human GVHD, we identify a new subset of postmitotic CD44(lo)CD62L(hi)CD8(+) T cells that generate and sustain all allogeneic T-cell subsets in GVHD reactions, including central memory, effector memory and effector CD8(+) T cells, while self-renewing. These cells express Sca-1, CD122 and Bcl-2, and induce GVHD upon transfer into secondary recipients. The postmitotic CD44(lo)CD62L(hi)CD8(+) T cells persist throughout the course of GVHD, are generated in the initial phase in response to alloantigens and dendritic cells and require <em>interleukin</em>-<em>15</em>. Thus, their long life, ability to self-renew and multipotentiality define these cells as candidate memory stem cells. Memory stem cells will be important targets for understanding and influencing diverse chronic immune reactions, including GVHD.

OBJECTIVE

This study investigates the course of serum cytokine levels in patients with multiple trauma, patients with a ruptured abdominal aortic aneurysm (AAA), and patients undergoing elective AAA repair and the relationship of these cytokines to the development of adult respiratory distress syndrome (ARDS) and multiple organ failure (MOF).

BACKGROUND

Severe tissue trauma, hemorrhagic shock, and ischemia-reperfusion injury are pathophysiologic mechanisms that may result in an excessive uncontrolled activation of inflammatory cells and mediators. This inflammatory response is thought to play a key role in the development of (remote) cell and organ dysfunction, which is the basis of ARDS and MOF.

METHODS

The study concerns 28 patients with multiple trauma, 20 patients admitted in shock because of a ruptured AAA, and 18 patients undergoing elective AAA repair. Arterial blood was serially sampled from admission (or at the start of elective operation) to day 13 in the intensive care unit, and the serum concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, and IL-6 were determined.

RESULTS

Twenty-two patients died, 15 within 48 hours and 7 after several weeks, as a result of ARDS/MOF. At hospital admission and after 6 hours, these nonsurvivors had significantly higher plasma TNF-alpha and IL-1 beta levels than did the survivors. At the same measuring points, TNF-alpha and IL-1 beta were significantly more elevated in patients with ruptured AAA than in traumatized patients. However, IL-6 was significantly higher in the traumatized patients. In 10 patients, ARDS/MOF developed, and 41 had an uncomplicated course in this respect. Those with ARDS/MOF exhibited significantly different cytokine patterns in the early postinjury phase. TNF-alpha and IL-1 beta levels were higher mainly on the first day of admission; IL-6 concentrations were significantly elevated in patients with ARDS/MOF from the second day onward. The latter cytokine showed a good correlation with the daily MOF score during the whole 2-week observation period.

CONCLUSIONS

In the early postinjury phase, higher concentrations of these cytokines are associated, not only with an increased mortality rate, but also with an increased risk for subsequent ARDS and MOF. These data therefore support the concept that these syndromes are caused by an overwhelming autodestructive inflammatory response.

Cytokines secreted by cells of the immune system can alter the behavior and properties of immune or other cells. At a site of inflammation, sets of cytokines interact with immune cells, and their combined effect is often more important than the function of one isolated component. Conventional techniques, such as enzyme-linked immunosorbent assays, generally require large quantities of cells to characterize a complete cytokine profile of activated lymphocytes. The Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology. We developed a multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen. Fifteen human cytokines (<em>interleukin</em> 1alpha [IL-1alpha], IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-<em>15</em>, IL-17, IL-18, gamma interferon, and tumor necrosis factor alpha) were validated with a panel of healthy individuals, rheumatoid arthritis patients, and juvenile idiopathic arthritis patients. Comparing the multiplex assay with a regular enzyme-linked immunosorbent assay technique with this donor panel resulted in correlation coefficients for all cytokines ranging from 0.75 to 0.99. Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.

Monocyte activation and adhesion to the endothelium play important roles in inflammatory and cardiovascular diseases. These processes are further aggravated by hyperglycemia, leading to cardiovascular complications in diabetes. We have previously shown that high glucose (HG) treatment activates monocytes and induces the expression of tumor necrosis factor (TNF)-alpha via oxidant stress and nuclear factor-kB transcription factor. To determine the effects of HG on the expression of other inflammatory genes, in the present study, HG-induced gene profiling was performed in THP-1 monocytes using cytokine gene arrays containing 375 known genes. HG treatment upregulated the expression of 41 genes and downregulated <em>15</em> genes that included chemokines, cytokines, chemokines receptors, adhesion molecules, and integrins. RT-PCR analysis further confirmed that HG significantly increased the expression of monocyte chemoattractant protein-1 (MCP-1), TNF-alpha, beta(2)-integrin, <em>interleukin</em>-1beta, and others. HG treatment increased transcription of the MCP-1 gene, MCP-1 protein levels, and adhesion of THP-1 cells to endothelial cells. HG-induced MCP-1 mRNA expression and monocyte adhesion were blocked by specific inhibitors of oxidant stress, protein kinase C, ERK1/2, and p38 mitogen-activated protein kinases. These results show for the first time that multiple inflammatory cytokines and chemokines relevant to the pathogenesis of diabetes complications are induced by HG via key signaling pathways.

Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN- dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of <em>interleukin</em> (IL)-<em>15</em> and IL-<em>15</em> receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.

The pool of memory T cells is regulated by homeostatic mechanisms to persist for prolonged periods at a relatively steady overall size. Recent work has shown that two members of the common gamma chain (gammac) family of cytokines, <em>interleukin</em>-7 (IL-7) and IL-<em>15</em>, govern homeostasis of memory T cells. These two cytokines work in conjunction to support memory T-cell survival and intermittent background proliferation. Normal animals contain significant numbers of spontaneously arising memory-phenotype (MP) cells, though whether these cells are representative of true antigen-specific memory T cells is unclear. Nevertheless, it appears that the two types of memory cells do not display identical homeostatic requirements. For antigen-specific memory CD8+ T cells, IL-7 is primarily important for survival while IL-<em>15</em> is crucial for their background proliferation. For memory CD4+ T cells, IL-7 has an important role, whereas the influence of IL-<em>15</em> is still unclear.

OBJECTIVE

To determine whether interleukin-1 (IL-1) or tumor necrosis factor alpha (TNFalpha), or both, plays a role in the excessive degradation that is observed in cultured osteoarthritic (OA) articular cartilage.

METHODS

Antagonists of IL-1 and TNFalpha, namely, IL-1 receptor antagonist and the PEGylated soluble TNFalpha receptor I, respectively, were added at different concentrations to explant cultures of nonarthritic (5 obtained at autopsy) and OA (15 obtained at arthroplasty) articular cartilage. The cleavage of type II collagen (CII) by collagenase was measured by an immunoassay in cartilage and culture media. Proteoglycan (mainly aggrecan) content and degradation were measured by a colorimetric assay for glycosaminoglycan (GAG) content in cartilage and culture media. Reverse transcriptase-polymerase chain reaction was used to analyze gene expression of matrix metalloproteases (MMPs) 1, 3, and 13, CII, aggrecan, IL-1, and TNFalpha.

RESULTS

Antagonists of IL-1 and TNFalpha inhibited the increase in CII cleavage by collagenase as well as the increase in GAG release observed in OA cartilage compared with normal cartilage. Inhibition was significant in tissue from some patients but not from others, although significant inhibition was observed when all the results were analyzed together. An increase in the GAG content in cartilage was seen in 4 of 15 cases. However, this increase was not significant when all the data were combined. Preliminary results indicated no effect of these antagonists on nonarthritic cartilage from 3 different donors. Independent analyses of gene expression in cultured cartilage from 9 other OA patients revealed that IL-1 or TNFalpha blockade, either alone and/or in combination, frequently down-regulated MMP-1, MMP-3, and MMP-13 expression. Expression of IL-1 and TNFalpha was inhibited by either antagonist or by the combination in essentially half the cases. The combined blockade up-regulated aggrecan and CII gene expression in approximately half the cases.

CONCLUSIONS

These results suggest that the autocrine/paracrine activities of TNFalpha and IL-1 in articular cartilage may play important roles in cartilage matrix degradation in OA patients but not in all patients. Inhibition of either or both of these cytokines may offer a useful therapeutic approach to the management of OA by reducing gene expression of MMPs involved in cartilage matrix degradation and favoring its repair.

Endotoxemia was evoked by bolus injection of Escherichia coli endotoxin (2 ng/kg body weight) in six healthy subjects to investigate the early kinetics of cytokine release in relation to the development of clinical and hematologic abnormalities frequently seen in gram-negative septicemia. The plasma concentration of tumor necrosis factor (TNF) increased markedly after 30 to 45 minutes, and reached a maximal level after 60 to 90 minutes. In each volunteer, the initial increase of plasma <em>interleukin</em> 6 (IL-6) concentrations occurred <em>15</em> minutes after the initial TNF increase, and maximal IL-6 concentrations were reached at 120 to <em>15</em>0 minutes. A transient increase in body temperature and pulse rate occurred simultaneously with the initial TNF and IL-6 increases, whereas a significant decrease in blood pressure occurred after 120 minutes. These changes were proportional to the changes in TNF and IL-6 concentrations. Coagulation activation, as assessed by a rise of prothrombin fragments and thrombin-antithrombin III complexes, was noted after 120 minutes, in the absence of activation of the contact system. A two- to sixfold increase in the concentrations of tissue plasminogen activator (t-PA) and von Willebrand factor antigen indicated endothelial cell activation. This increase started at 120 and 90 minutes, respectively. The release of t-PA coincided with activation of the fibrinolytic pathway, as measured by plasmin-alpha 2-antiplasmin complexes. The fibrinolytic activity of t-PA was subsequently offset by release of plasminogen activator inhibitor, observed <em>15</em>0 minutes after the endotoxin injection, and reaching a peak at 240 minutes. No complement activation was detected. These results show that in humans endotoxin induces an early, rapidly counteracted fibrinolytic response, and a more long-lasting activation of thrombin by a mechanism other than contact system activation. In addition, our data suggest that endotoxin-induced leukopenia and endothelial cell activation are mediated by TNF.

CD94/NKG2C(+) natural killer (NK) cells are increased in healthy individuals infected with human cytomegalovirus (HCMV), suggesting that HCMV infection may shape the NK cell receptor repertoire. To address this question, we analyzed the distribution of NK cell subsets in peripheral blood lymphocytes (PBLs) cocultured with HCMV-infected fibroblasts. A substantial increase of NK cells was detected by day 10 in samples from a group of HCMV(+) donors, and CD94/NKG2C(+) cells outnumbered the CD94/NKG2A(+) subset. Fibroblast infection was required to induce the preferential expansion of CD94/NKG2C(+) NK cells that was comparable with allogeneic or autologous fibroblasts, and different virus strains. A CD94-specific monoclonal antibody (mAb) abrogated the effect, supporting an involvement of the lectinlike receptor. Purified CD56(+) populations stimulated with HCMV-infected cells did not proliferate, but the expansion of the CD94/NKG2C(+) subset was detected in the presence of <em>interleukin</em>-<em>15</em> (IL-<em>15</em>). Experiments with HCMV deletion mutants indicated that the response of CD94/NKG2C(+) NK cells was independent of the UL16, UL18, and UL40 HCMV genes, but was impaired when cells were infected with a mutant lacking the US2-11 gene region. Taken together the data support that the interaction of CD94/NKG2C with HCMV-infected fibroblasts, concomitant to the inhibition of human leukocyte antigen (HLA) class I expression, promotes an outgrowth of CD94/NKG2C(+) NK cells.

Infection with hepatitis C virus (HCV) is a leading cause of chronic liver disease worldwide. Little is known about how this virus is able to persist or whether this persistence might be because of its ability to alter the early innate immune response. The major HCV envelope protein E2 has been shown to bind to CD81. Thus, HCV binding to natural killer (NK) cells could result in the cross-linking of CD81. To explore this possibility, we investigated whether cross-linking CD81 on NK cells could alter NK cell function. CD81 cross-linking by monoclonal antibody (mAb) specific for CD81 or by immobilized E2 have been shown to result in costimulatory signals for human T cells. In this study, we show that CD81 cross-linking via immobilized E2 or mAbs specific for CD81 inhibits not only non major histocompatibility complex-restricted cytotoxicity mediated by NK cells but also interferon (IFN)-gamma production by NK cells after exposure to <em>interleukin</em> (IL)-2, IL-12, IL-<em>15</em>, or CD16 cross-linking. These results show that CD81 cross-linking mediates completely different signals in NK cells versus T cells. Importantly, these results suggest that one mechanism whereby HCV can alter host defenses and innate immunity is via the early inhibition of IFN-gamma production by NK cells.

To infer possible mechanisms of acute airway inflammation and mucus hypersecretion in acute severe asthma, we performed cellular and biochemical analysis on sputum from 18 adults with acute severe asthma and compared the results with results of analysis of sputum from 12 adults with cystic fibrosis (CF). We found that in subjects with asthma neutrophils made up more than 75% of sputum cells in 10 samples whereas eosinophils made up more than 75% of cells in only three samples. Fifty percent of the subjects with asthma reported that their asthma exacerbation was precipitated by a respiratory tract infection, and these subjects had a significantly higher percentage of neutrophils in their sputum (85% +/- 6% vs 57% +/- 12%, p = 0.05). In the CF samples neutrophils made up more than 95% and eosinophils less than 1% of cells in all samples analyzed. Analysis of fluid phase chemicals in asthmatic and CF sputum samples showed that despite overall lower mean values of neutrophil elastase (27 +/- 11 micrograms/ml vs 466 +/- 121 micrograms/ml, p = 0.0001) and <em>interleukin</em>-8 (IL-8) (55 +/- <em>15</em> ng/ml vs 186 +/- 24 ng/ml, p = 0.0001), some of the asthmatic samples had values for these variables that overlapped those in the CF samples. In addition, the asthmatic samples were distinguished by the presence of higher tryptase (10 +/- 7 U/L vs 0.9 +/- 0.9 U/L, p = 0.0001) and <em>interleukin</em>-6 (1166 +/- 447 ng/ml vs 186 +/- 24 ng/ml; p = 0.0001) levels and by a higher ratio of albumin to mucin-like glycoprotein (0.8 +/- 0.5 vs 0.1 +/- 0.002, p = 0.02). DNA levels were lower in the asthmatic samples (0.5 +/- 0.3 mg/ml vs 3.5 +/- 1.2 mg/ml, p = 0.05). We conclude that neutrophils predominate more frequently than eosinophils as the major inflammatory cell in sputum from patients with asthma in acute exacerbation. We speculate that this may be because respiratory tract infections are a frequent precipitant of acute asthma. In addition, the high IL-8 levels and free neutrophil elastase activity observed in asthmatic sputum suggests that IL-8 may mediate airway neutrophilia in acute asthma and that neutrophil elastase may mediate mucin glycoprotein hypersecretion in acute asthma, as has been proposed for the mucin hypersecretion in CF.