TL1A/DR3 axis involvement in the inflammatory cytokine network during pulmonary sarcoidosis.

BACKGROUND: TNF-like ligand 1A (TL1A), a recently recognized member of the TNF superfamily, and its death domain receptor 3 (DR3), firstly identified for their relevant role in T lymphocyte homeostasis, are now well-known mediators of several immune-inflammatory diseases, ranging from rheumatoid arthritis to inflammatory bowel diseases to psoriasis, whereas no data are available on their involvement in sarcoidosis, a multisystemic granulomatous disease where a deregulated T helper (Th)1/Th17 response takes place. METHODS: In this study, by flow cytometry, real-time PCR, confocal microscopy and immunohistochemistry analyses, TL1A and DR3 were investigated in the pulmonary cells and the peripheral blood of 43 patients affected by sarcoidosis in different phases of the disease (29 patients with active sarcoidosis, 14 with the inactive form) and in 8 control subjects. RESULTS: Our results demonstrated a significant higher expression, both at protein and mRNA levels, of TL1A and DR3 in pulmonary T cells and alveolar macrophages of patients with active sarcoidosis as compared to patients with the inactive form of the disease and to controls. In patients with sarcoidosis TL1A was strongly more expressed in the lung than the blood, i.e., at the site of the involved organ. Additionally, zymography assays showed that TL1A is able to increase the production of matrix metalloproteinase 9 by sarcoid alveolar macrophages characterized, in patients with the active form of the disease, by reduced mRNA levels of the tissue inhibitor of metalloproteinase (TIMP)-1. CONCLUSIONS: These data suggest that TL1A/DR3 interactions are part of the extended and complex immune-inflammatory network that characterizes sarcoidosis during its active phase and may contribute to the pathogenesis and to the progression of the disease.

Patients with primary antiphospholipid antibody syndrome and without associated vascular risk factors present a normal endothelial function.

INTRODUCTION: Primary antiphospholipid antibody syndrome (PAPS) is characterized by venous or arterial thrombosis and positive antiphospholipid antibodies. It is controversial whether PAPS patients have early atherosclerosis. Endothelial dysfunction is an early event in the natural history of atherosclerosis. Aim of our study was to compare endothelial function of patients with PAPS and no associated risk factors with that of age- and sex-matched controls. MATERIALS AND METHODS: Patients with PAPS, carefully selected to exclude all known risk factors for cardiovascular diseases, estrogen therapy, pregnancy, intake of drugs affecting endothelial function, vitamins or antioxidants, were included in a case-control study. Controls were age- (+/-5 years) and sex-matched subjects with the same exclusion criteria but without PAPS. Flow-mediated dilation of the brachial artery and some plasmatic markers of endothelial and platelet activation were measured. Measures are expressed as mean+/-SEM. RESULTS: Twenty cases (mean age 42+/-4.0 years, 11 females) and 39 controls (mean age 41+/-2.9, 22 females) were studied. FMD was 5.7+/-0.8% in cases (95% CI: 4.1 to 7.3) and 6.8+/-0.5% (5.7 to 7.9) in controls (p=NS). Plasma von Willebrand factor was 128+/-11.3% and 134.2+/-16.1% in cases and controls, respectively (p=NS). Soluble P-selectin and soluble CD40L were 94.1+/-4.9 ng/ml and 0.7+/-0.1 ng/ml in cases and 87.7+/-4.0 ng/ml and 1.0+/-0.2 in controls, respectively (p=NS). In a substudy, circulating progenitor and mature endothelial cells were comparable between the two groups. CONCLUSIONS: Endothelial function in patients with PAPS and no associated risk factors is similar to that of age- and sex- matched controls. These data suggest that the alterations leading to thrombosis in PAPS concern primarily the clotting system.

Relationship between stress hormones and immune response during high-altitude exposure in women.

We previously confirmed that high altitude (HA) exposure can modify the number and function of immune cells, leading to a disruption in the homeostatic regulation of T helper1 (Th1)/T helper2 (Th2) immune responses. Our aim was to evaluate possible relationships between the stress response and immunological parameters during HA exposure. Thirteen healthy women spent 21 days at 5050 m. Before (SL1), the first and the 21st day at HA (HA1 and HA2, respectively), and the day after returning at sea level (SL2), we collected blood samples for immunologic parameters, and 24-h urine samples for norepinephrine, epinephrine, and cortisol. Norepinephrine and cortisol significantly increased (p<0.01) at HA1 and HA2 compared to SL1, while epinephrine did not change. At HA1, CD3+ T-cell fell significantly (p<0.001) with respect to SL1, owing to a significant (p<0.001) CD4+ T-cell reduction, while CD16+ and CD56+ increased (p<0.001) at HA2 compared to SL1. The expression of interferon-gamma (IFN-gamma) decreased (p<0.0005) at HA1 and HA2 with respect to SL1. At HA1 different lymphocyte subset (CD3+, CD4+, CD19+) were well correlated with epinephrine (p<0.05), whereas in analyzing the combined data (SL1-HA1-HA2-SL2), CD3+ (r=-0.310), CD4+ (r=-0.332), CD16+ (r=0.404), and CD56+ (r=0.373) demonstrated moderate but significant correlations (p<0.05) with norepinephrine. Moreover, norepinephrine levels were inversely correlated (r=-0.591; p<0.001) with IFN-gamma expression, a typical Th1 cytokine. We suggest that the sympatho-adrenal axis may have a role on the immunologic adaptations observed during HA exposure, and specifically on the observed impairment of the Th1/Th2 immune balance.

Genotypic evaluation of killer immunoglobulin-like receptors in NK-type lymphoproliferative disease of granular lymphocytes.

Using polymerase chain reaction (PCR)-based sequence-specific primers, the killer immunoglobulin-like receptor (KIR) genotypes of 35 patients with natural killer (NK)-type lymphoproliferative disease of granular lymphocytes and of 50 normal subjects were investigated to evaluate whether genes coding for activating KIRs were more frequently detected in patients with NK-lymphoproliferative disease of granular lymphocytes (LDGL). Genotype frequency indicated that the most frequently found gene content was eight genes in controls and 14 in patients (P<0.05). The KIR genotype analysis revealed that patient and, surprisingly, control KIR genotypes preferentially consisted of type B haplotypes characterized by the presence of multiple-activating KIRs. Evidence was also provided that the same KIR genotype was shared by a variable number of patients. Interestingly, the recurrent genotypes observed in the patient group were not found in controls. Concerning inhibitory genes, KIR2DL5a and 2DL5b were more frequently detected in patients than in controls (P<0.01), likely representing a discrete feature of the genetic repertoire of the patients. KIR gene repertoire analysis in patients suggests that the susceptibility to NK-LDGL might be related to the presence of activating KIR genes and supports the concept that these receptors may be involved in the priming of granular lymphocytes (GL) proliferation. Population analysis might disclose a genetic background predisposing to this disease.

The chemokine receptor CXCR3 is expressed on malignant B cells and mediates chemotaxis.

B- and T-cell recirculation is crucial for the function of the immune system, with the control of cell migration being mainly mediated by several chemokines and their receptors. In this study, we investigated the expression and function of CXCR3 on normal and malignant B cells from 65 patients with chronic lymphoproliferative disorders (CLDs). Although CXCR3 is lacking on CD5(+) and CD5(-) B cells from healthy subjects, it is expressed on leukemic B lymphocytes from all (31/31) patients with chronic lymphocytic leukemia (CLL). The presence of CXCR3 was heterogeneous in other B-cell disorders, being expressed in 2 of 7 patients with mantle cell lymphoma (MCL), 4 of 12 patients with hairy cell leukemia (HCL), and 11 of 15 patients with other subtypes of non-Hodgkin's lymphomas (NHLs). Chemotaxis assay shows that normal B cells from healthy subjects do not migrate in response to IFN-inducible protein 10 (IP-10) and IFN-gamma-induced monokine (Mig). In contrast, a definite migration in response to IP-10 and Mig has been observed in all malignant B cells from patients with CLL, but not in patients with HCL or MCL (1/7 cases tested). Neoplastic B cells from other NHLs showed a heterogenous pattern. The migration elicited by IP-10 and Mig was inhibited by blocking CXCR3. No effect of IP-10 and Mig chemokines was observed on the cytosolic calcium concentration in malignant B cells. The data reported here demonstrate that CXCR3 is expressed on malignant B cells from CLDs, particularly in patients with CLL, and represents a fully functional receptor involved in chemotaxis of malignant B lymphocytes.

B lymphocytes from patients with chronic lymphoproliferative disorders are equipped with different costimulatory molecules.

Several costimulatory molecules play a key role in the differentiation of B lymphocytes and in T-B-cell interactions. In this study, we addressed the question of whether different receptors and counter-receptors may be expressed on malignant B lymphocytes from chronic B-cell malignancies. Using flow cytometry and reverse transcription PCR analyses, the expression of molecules belonging to the tumor necrosis factor receptor (TNFR) and tumor necrosis factor ligand (TNFL) families, as well as the expression of CD80 and CD86 molecules, was analyzed in normal B cells and in different chronic lymphoproliferative disorders of B-cell type, including B-cell chronic lymphocytic leukemia (CLL), mantle cell lymphoma, hairy cell leukemia (HCL), and HCL variant. Different patterns of expression of TNFR and TNFL superfamily molecules were demonstrated among B-cell malignancies. In particular, CD40 was commonly observed on all B cells (both tumor and normal), whereas its ligand (CD40L), which is usually undetectable on resting normal B lymphocytes, was expressed in CLL and HCL but not in other chronic lymphoproliferative disorders. CD27 was not shown in normal B cells, although it was present in all malignancies and with particularly high density in mantle cell lymphoma. CD70 was widely distributed on tumor B lymphocytes, but not on the CD5+ normal counterpart. CD30 was strongly expressed in HCL variant and weakly in B-cell CLL, whereas its ligand showed a wide pattern of expression, including all neoplastic and normal B cells. TNFR II (CD120b) and CD80 were distributed on neoplastic B cells from all groups, usually at an intermediate to high degree of intensity, whereas the CD86 molecule was present at lower intensity than CD80. Finally, reverse transcription PCR analysis confirmed the presence of CD40L, CD30, and CD30L mRNAs in those B cells expressing the corresponding membrane-bound proteins at low density. Our data indicate that TNFR and TNFL molecules are of use clinically both in differentiating B-cell malignancies from the normal counterpart (i.e., CD27, CD70, CD40L, CD30, and CD80) and in defining different chronic B-cell disorders (i.e., CD40L, CD27, and CD30). Interestingly, the observation that several receptors and their ligands (i.e., CD40/CD40L, CD30/CD30L, and CD27/CD70) can be expressed on the same cell suggests that these molecules play a role in initiating and maintaining the neoplastic process by mediating B-T and B-B interactions.

Analysis of the T cell receptor in the lymphoproliferative disease of granular lymphocytes: superantigen activation of clonal CD3+ granular lymphocytes.

To investigate whether cell populations in CD3+ lymphoproliferative disease of granular lymphocytes (LDGLs) were skewed toward the use of specific V beta regions, we studied the repertoire of T-cell receptor (TCR) V beta gene products in 18 patients, as well as their relationship to the clonal bands in the Southern blot and the activation mediated by superantigens. Using a panel of monoclonal antibodies (mAbs) for conserved V beta segments and PCR, a dominant population expressing a specific V beta region was demonstrated in all patients. In five (27%) cases, granular lymphocytes (GLs) were found to express the V beta 13.1, while V beta 8 and V beta 6 were each expressed in three (17%) cases. The remaining cases were characterized by the proliferation of TCR V beta 2, V beta 3, V beta 4, V beta 9, V beta 12, V beta 16, and V beta 20. This finding indicates a biased usage of a limited TCR V beta in LDGLs, since nearly 60% of the cases utilized only three families of the TCR V beta genes. In all of the cases studied, we proved that the subset recognized by mAb and PCR was identical to that accounting for the extra band(s) of the Southern blot. This finding confirms the clonal nature of the population identified according to TCR V beta expression both by phenotype and PCR. On functional grounds, we evaluated whether GLs can be activated through the specific TCR using the superantigens recognizing discrete V beta families, such as staphylococcal proteins, including SEA, SEB, SEC1, SEC2, SED, and SEE. We demonstrated that the TCR-alpha/beta of clonal GLs in LDGL patients is functionally active in delivering cytotoxic and proliferative signals upon superantigen activation.

Serologic and molecular evidence for a possible pathogenetic role of viral infection in CD3-negative natural killer-type lymphoproliferative disease of granular lymphocytes.

We studied a series of 18 patients with CD3- lymphoproliferative disease of granular lymphocytes (LDGL) for evidence of chronic viral infection, including Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human T lymphotropic virus (HTLV), and human immunodeficiency virus (HIV). Although all patients tested had serologic evidence for past infection with EBV, polymerase chain reaction (PCR) analysis of peripheral blood mononuclear cell (PBMC) DNA utilizing specific EBV primers demonstrated the presence of EBV-DNA in only six of 17 CD3- LDGL cases. A previous history of HBV infection, as defined by the presence of circulating IgG anti-HBc antibodies associated with either HBsAg positivity or negativity, was documented in seven cases; however, viral DNA was not detected in PBMC of these patients using PCR with specific HBV primers. Specific anti-HCV antibodies, confirmed by recombinant immunoblot assay, were detected in five CD3- LDGL patients; PCR analysis demonstrated the presence of viral RNA in PBMC of two of these cases. No patient had antibodies to HTLV-I/II or HIV-1/2. Five patients were infected by more than one virus (two with HBV and EBV and three with HBV and HCV). Our results provide serologic evidence for past viral infection in the large majority of CD3- NK-type LDGL patients. These data suggest that viral infection may have played a role early in disease pathogenesis and may no longer be necessary in sustaining GL proliferation in CD3- NK-type LDGL.

Skewing of the T-cell receptor repertoire in the lung of patients with HIV-1 infection.

OBJECTIVES: A bias of the use of T-cell receptor (TCR) V beta regions has been reported both in peripheral blood and in several tissues in patients with AIDS, including lymph nodes, spleen and salivary glands. Although the disease is frequently characterized by an infiltration of T cells in the lung interstitium, no information is presently available on the configuration of the TCR repertoire in this microenvironment. This study was performed to address the question of whether a bias in T-cell selection occurs in the lung of patients with AIDS. METHODS: TCR beta-chain variable region (TCR-V beta) repertoire was analysed by flow cytometry and polymerase chain reaction (PCR) analyses in blood and lung lymphocytes of 13 patients with HIV-1 infection at different stages of the disease. Blood and lung lymphocytes were also assessed for their responsiveness to different superantigenic stimuli represented by staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SED and SEE). RESULTS: Flow cytometry analysis in AIDS patients demonstrated an overexpression of cells bearing V beta 2 and V beta 3 gene segments in the lung compared with peripheral blood of the same subjects, as well as to lung and blood lymphocytes of normal controls. PCR analysis performed in AIDS patients extended these observations and demonstrated a significant bias also in the use of T cells bearing V beta 7 and V beta 9 gene regions in the lung compartment with respect to the blood. Virtually all T cells bearing the overrepresented V beta segment belong to the CD8 subset. Interestingly, T-lymphocyte response to different superantigens demonstrated a low proliferative rate in the lung with respect to the blood in HIV-1-infected patients. CONCLUSIONS: These findings indicate a compartmentalization of cells bearing discrete V beta gene products in the pulmonary microenvironment of patients with AIDS and suggest that the expansion of specific-V beta region subsets occurring in the lung might result from triggering by a superantigen.

Demonstration of Chlamydia pneumoniae in atherosclerotic arteries from various vascular regions.

Chlamydia pneumoniae (CP) has been reported to be a pathogenic agent in the mechanism leading to atherosclerosis. The majority of available data is focused mainly on coronary artery disease whereas the distribution of CP in different areas, associated with atherosclerotic disorders, has not been completely clarified. In this study we investigated the presence of CP in atheromasic plaques from five different vascular areas (basilary artery, coronary artery, thoracic aorta, abdominal aorta, renal arteries) using nested polymerase chain reaction (PCR) and immunohistochemical staining (IHC), in order to establish the putative association of CP with atherosclerotic disease. The same atheromasic plaques were also tested for the presence of Helicobacter pylori (HP) and cytomegalovirus (CMV), other putative agents of atherosclerosis, using a nested PCR technique. Our data indicate that the presence of CP can be demonstrated in 100% of patients tested, considering globally the five areas of analysis. On the other hand the presence of HP has been demonstrated in four out of 18 patients (22.2%), and CMV only in three out of 18 (16.6%). Our results strongly suggest an association between CP and atherosclerosis and highlight the need for the detection of CP in multiple vascular areas of the same patient.

Alveolar macrophage-T cell interactions during Th1-type sarcoid inflammation.

Sarcoidosis is an immunomediated, multisystem disorder of unknown cause(s) characterized by a heightened Th1 immune response that leads to an uncontrolled granuloma formation at sites of disease activity. The past few years have seen outstanding advances in the understanding of immunological and molecular events involved in the pathogenesis of this disease. The idea is that several cytokines and chemokines, which are secreted at sites of disease activity, participate in granuloma formation. This paper describes recent data that have clarified some of the events that govern the development of the hypersensitivity reaction during sarcoidosis. In particular, we will review recent evidence indicating that a complex relationship exists between the macrophage/lymphocyte cellular axis and the tissue networks of cytokines.

Interleukin-15: a novel cytokine with regulatory properties on normal and neoplastic B lymphocytes.

IL-15 is a recently discovered cytokine that shares biological activities with IL-2. Although the biological functions displayed by these two molecules overlap to some extent, they are produced by different cell types and bind to distinct receptorial structures. Both cytokines transduce signals through the beta (p75) and gamma (p64) chains of the IL-2R system, but IL-15, like IL-2, binds to its own specific alpha chain, referred to as IL-15Ralpha. Similarly to IL-2, IL-15 is able to trigger both the proliferation and immunoglobulin production by normal B-lymphocytes. These biological functions may be acquired however only when B-cells have been preactivated in vitro with polyclonal mitogens, or alternatively, when they are cultured in association with other stimuli. By contrast, leukemic cells from patients with chronic B-cell malignancies, including B-cell chronic lymphocytic leukemia and hairy cell leukemia, proliferate to IL-15 regardless of in vitro preactivation. This peculiar IL-15 responsiveness distinguishes malignant B-cells from normal B-lymphocytes. Furthermore, the proliferation elicited by IL-15 in B-CLL and HCL is mainly related to the presence of the beta and gamma chains of the IL-2R system on malignant B-lymphocytes.

Competitive binding assay for quantitative determination of GM1 ganglioside in plasma and cerebrospinal fluid.

A competitive binding assay for the quantitative determination of GM1 ganglioside is described. After extraction from biological fluids, GM1 was incubated with a known amount of cholera toxin B-subunit conjugated with horseradish peroxidase, and exposed to GM1 adsorbed onto polystyrene microwells. Since GM1 in solution blocks the binding of toxin B-subunit to GM1 adsorbed onto the solid phase, enzyme activity serves as a reciprocal measure of GM1 concentration in the sample. The assay was used to determine the basal level of GM1 in plasma and cerebrospinal fluid in different populations.

Lung lymphocytes: origin, biological functions, and laboratory techniques for their study in immune-mediated pulmonary disorders.

Different types of immunocompetent cells, including T lymphocytes and alveolar macrophages, account for pulmonary host defense. Taking advantage of the availability of the monoclonal antibody technique, cell culture facilities, pure recombinant cytokines, and molecular probes for their genes, in the last few years it has been possible to keenly study the different steps that lead to the compartmentalization of immune response in human lung. Furthermore, the immunological analysis of cells retrieved from bronchoalveolar lavage (BAL) allowed recognition of the importance of immune mechanisms in the evolution of immune-mediated pulmonary disorders. The purpose of this review is to summarize recent advances on the immunologic characterization of lung lymphocytes in health and disease. Following a brief description of the pathways through which the pulmonary lymphoid system contributes to removing potentially harmful inhaled antigenic materials, available laboratory techniques to evaluate the lymphoid component of the pulmonary immune system and their byproducts are discussed. These techniques cover methods for preparing lymphocytes from the BAL fluid and for characterizing lung lymphocytes both in cell suspensions and pulmonary tissue biopsies. Other sections of this review describe the techniques for measuring the immunologic effector functions of lung lymphocytes. We also provide the reader with a flavor of the molecular biology methods used to characterize lymphocytes in the pulmonary microenvironment. The final sections of the review article highlight the pathogenetic role envisaged for lymphoid cells in pulmonary disease states and emphasize the importance of the BAL analysis in the clinical management of the most relevant immune-mediated lung disease.

Analysis of TNF-receptor and ligand superfamily molecules in patients with lymphoproliferative disease of granular lymphocytes.

In 21 patients with lymphoproliferative disease of granular lymphocytes (LDGL), we investigated the expression and the function of molecules belonging to TNF-receptor and TNF-ligand superfamilies (CD30/CD30L; CD40/CD40L; CD27/CD70; Fas [CD95]/FasL[CD95L]). Fourteen patients were characterized by a proliferation of granular lymphocytes (GLs) expressing the CD3(+)CD16(+) phenotype, whereas 7 cases showed the CD3(-)CD16(+) CD56 +/- phenotype. Our data show that both CD3(+) and CD3-GLs are preferentially equipped with CD30, CD40, CD40L, CD70, and CD95 antigens; this pattern is usually associated with the lack of CD27 and CD30L antigens expression. CD95L was demonstrated in the cytoplasm in 14 of 21 cases by flow cytometry, but a definite signal was demonstrated in all cases studied using polymerase chain reaction analysis. On functional grounds, a stimulatory activity on rIL-2 mediated redirected-cytotoxicity against Fcgamma+ P815 targets was demonstrated with anti-CD30, CD40, CD40L, CD70, CD95, and CD95L mAbs, although resting cells were unable to exhibit significant redirected-cell lysis. The addition of anti-CD30, CD30L, CD40, CD40L, CD95, and CD95L mAbs did not show any significant effect on cell proliferation at resting conditions or after rIL-2 stimulation, whereas anti-CD70 mAb mediated cell proliferation in 6 of 10 cases tested. This figure was not related to an increase in apoptotic cells, as investigated by Annexin-V expression. Our data indicate that both CD3(+) and CD3(-) GLs are equipped with different costimulatory antigens, supporting the concept that these cells are in vivo activated and suggesting that these molecules might play a role in the cytotoxic mechanisms of GLs. (Blood. 2000;96:647-654)

Spontaneous resolution of p58/EB6 antigen restricted NK-type lymphoproliferative disease of granular lymphocytes: role of Epstein Barr virus infection.

We describe a patient with a CD3- lymphoproliferative disease of granular lymphocytes (LDGL) characterized by proliferation of CD3-CD16+ GL, restricted to the expression of p58/EB6 antigen and lacking the p58/GL183 antigen. Using PCR analysis we demonstrated the presence of EBV DNA in the peripheral blood mononuclear cells and purified CD16+ GL from the patient; a monoclonal episomic configuration of the virus could not be demonstrated with Southern blot analysis. The presence of EBV DNA was also detected by PCR in the serum; this finding was associated with a serological pattern consistent with a previous, already seroconverted, EBV infection. During a 4-year follow-up the lymphocytosis spontaneously disappeared; interestingly, in terms of the p58 antigen expression, we provided evidence of the reconstitution of a normal pattern of circulating NK subsets (i.e. p58/EB6+ p58/GL183-, p58/EB6+ p58/GL183+, p58/EB6- p58/GL183-, p58/EB6-p58/GL183+). At the time of resolution of lymphocytosis, EBV-PCR analysis still demonstrated the persistence of EBV DNA in peripheral blood mononuclear cells, but not in the patient's serum. By indicating that inciting agents (in this case EBV) are involved in inducing the GL proliferation, our data contribute insights into the pathogenetic mechanisms accounting for in vivo GL accumulation in LDGL. It appears that a second, still unknown, event is required to determine the neoplastic transformation.

Expression of tumor necrosis factor-receptor superfamily members by lung T lymphocytes in interstitial lung disease.

Recently, a novel receptor superfamily has been identified whose members interact with a parallel family of ligands showing homology to tumor necrosis factor (TNF). To investigate the role of these receptor structures in the pulmonary environment, we evaluated the expression of some members of the TNF-receptor (CD27, CD30, CD40, CD95/Fas, CD120a, and CD120b) and TNF-ligand (CD40L, CD70/CD27L, CD30L, and mTNFalpha) superfamilies by bronchoalveolar lavage (BAL) T cells recovered from healthy subjects and patients with interstitial lung disease (ILD). Lung T lymphocytes recovered from control subjects showed a slight expression of CD27 but did not bear CD30, CD40, CD120a, or CD120b antigens. CD27 expression was restricted to normal CD4+ cells. Fas antigen (CD95), which is involved in activation-driven T-cell suicide, and the ligand for CD27 (CD70) were weakly expressed by normal BAL T-cell subpopulations. In patients with sarcoidosis, the majority of pulmonary T lymphocytes were CD4+ cells that expressed low levels of CD27 antigen and an upregulation of CD95 and CD70 molecules. When we characterized lymphocytes accumulating in the lung of patients with HIV infection and hypersensitivity pneumonitis, we demonstrated that T cells accounting for the CD8 alveolitis bore TNF-receptor type 2 (CD120b) at high density and were CD70+ while CD40L, CD30L, or mTNF-alpha expression were not found. The discrete surface expression of the TNF-receptors and TNF-ligands on alveolar T-cell subsets suggests that these molecules play a role in the immune regulatory mechanisms that ultimately lead to the alveolitis in the pulmonary microenvironment of interstitial lung disease.

Interleukin-15 triggers activation and growth of the CD8 T-cell pool in extravascular tissues of patients with acquired immunodeficiency syndrome.

The impairment of interleukin-2 (IL-2) production occurs very early after human immunodeficiency virus (HIV) infection as a consequence of the quantitative depletion of Th1 cells. Despite the shift in cytokine production, most individuals develop an oligoclonal expansion of major histocompatibility complex restricted, HIV-specific CD8+ cytotoxic T lymphocytes (CTL) in different organs, suggesting that other cytokines replace IL-2 in initiating the tissue infiltration of CD8+ T cells. In this study we show that IL-15, a product of monocyte-macrophages and non-T cells and which has overlapping biological activities with IL-2, is involved in local cell networks accounting for the activation and expansion of CD8+ T-cell pools in a highly affected organ, ie, the lung. IL-15 induced proliferation of T cells obtained from the lower respiratory tract of HIV-infected patients with T-cell alveolitis and severe depletion of CD4+ T cells. Lung lymphocytes were CD45R0+/CD8+ T cells spontaneously expressing activation markers (CD69 and HLA-DR) and equipped with the receptorial subunits which bind IL-15, notably the beta and gamma chains of the IL-2 receptor (IL-2R) and the recently identified IL-15 binding-protein termed IL-15R alpha. Similar phenotypic findings were obtained after incubation of normal T cells with IL-15, which induced CD8+ T cells to express activation markers and to proliferate. The block of the IL-2R beta/IL-2R gamma complex with specific monoclonal antibodies abolished the T-cell stimulatory activity of IL-15 while the combination of IL-15 and tumor necrosis factor-alpha upregulated the proliferative response of lung T lymphocytes. The hypothesis that the tissue growth of lung CD8+ lymphocytes may involve cytokines produced from cells other than T lymphocytes was confirmed by the evidence that pulmonary macrophages expressed high levels of IL-15 and that anti-IL-15 antibodies inhibited the accessory function of alveolar macrophages on mitogen-induced CD8+ T-cell proliferation. Together, these results suggest that macrophage-derived cytokines produced at sites of T-cell infiltration play a role in the activation of HIV-specific CD8+ T-cell-mediated immune response.

CD8 T-cell infiltration in extravascular tissues of patients with human immunodeficiency virus infection. Interleukin-15 upmodulates costimulatory pathways involved in the antigen-presenting cells-T-cell interaction.

Interleukin (IL)-15 regulates the proliferative activity of the CD8(+) T-cell pool in human immunodeficiency virus (HIV)-infected patients, thereby contributing to the maintenance of the CD8(+) T-cell-mediated immune response against HIV in extravascular tissues, including the lung. However, the effects of IL-15 on antigen-presenting cells (APC) during HIV infection are still unclear. In this study, we evaluated whether IL-15 regulates the macrophage stimulatory pathways governing inflammatory events that take place in the lung of patients with HIV infection. As a first step we evaluated the in vitro effects of IL-15 on lung macrophages retrieved from the respiratory tract of eight normal subjects. Although macrophages from uninfected individuals expressed the IL-15 binding proteins (IL-15Ralpha and the common gammac) at resting conditions, they did not express IL-15 messenger RNA (mRNA). However, a 24-hour stimulation with IL-15 induced the expression of interferon-gamma (IFN-gamma) and IL-15 itself, suggesting a role for this cytokine in the activation of the pulmonary macrophage pool during inflammation. As a confirmation of the role of IL-15 in this setting, at resting conditions, alveolar macrophages of patients with HIV infection and T-cell alveolitis expressed IL-15, IFN-gamma, and IL-15 binding proteins; showed an upmodulation of costimulatory molecules, B7 and CD72, which are involved in the APC of macrophages; and behaved as effective accessory cells because they elicited a strong proliferation of T cells. The accessory effect was inhibited by pretreatment with anti-CD72, anti-B7 (CD80 and CD86), and anti-IL-15 monoclonal antibodies (MoAb). We then investigated the relationship between IL-15 and the expression of costimulatory molecules by macrophages. A 24-hour stimulation of IL-15Ralpha+/gammac+ macrophages with IL-15 upregulated the expression of CD80 and CD86. The evidence that IL-15 upregulates the expression of coligands that favor the contact between T cells and APC, per se, triggers T-cell activation and proliferation and acts as a chemoattractant for T cells, suggests that IL-15 plays a key role in Tc1-mediated defense mechanisms taking place in extravascular tissues of patients with HIV disease.

Regulation of alveolar macrophage-T cell interactions during Th1-type sarcoid inflammatory process.

The accessory function of antigen-presenting cells depends on the presence of a number of costimulatory molecules, including members of the B7 family (CD80 and CD86) and the CD5 coligand CD72. The aim of this study was to evaluate the regulation of T cell-antigen-presenting cell costimulatory pathways in the lung of patients with a typical Th1-type reaction, i.e., sarcoidosis. Although normal alveolar macrophages (AMs) did not bear or bore low levels of costimulatory molecules, AMs from sarcoid patients with CD4 T-cell alveolitis upmodulated CD80, CD86, and CD72 and expressed high levels of interleukin (IL)-15; lymphocytes accounting for T-cell alveolitis expressed Th1-type cytokines [interferon (IFN)-gamma and/or IL-2] and bore high levels of CD5 and CD28 but not of CD152 molecules. In vitro stimulation of AMs with Th1-related cytokines (IL-15 and IFN-gamma) upregulated the expression of CD80 and CD86 molecules. However, stimulation with IL-15 induced the expression of Th1-type cytokines (IFN-gamma) and CD28 on sarcoid T cells, suggesting a role for this macrophage-derived cytokine in the activation of the sarcoid T-cell pool. The hypothesis that CD80 and CD86 molecules regulate the sarcoid T-cell response was confirmed by the evidence that AMs induced a strong proliferation of T cells that was inhibited by pretreatment with CD80 and CD86 monoclonal antibodies. To account for these data, it is proposed that locally released cytokines provide AMs with accessory properties that contribute to the development of sarcoid T-cell alveolitis.