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.

Apoptotic effect of cyclosporin a and dexamethasone in malignant cells of patients with B-chronic lymphocytic leukemia.

B-chronic lymphocytic leukemia (B-CLL) is a malignant disorder characterized by the accumulation of the leukemic cells in the G0-G1 phase of the cell cycle and expressing high levels of the anti-apoptotic protein Bcl-2. Since we observed that the treatment of autoimmune complications with Cyclosporine A (CsA) determined in some CLL patients an improvement not only of the autoimmune phenomena, but also of the leukemic process, we evaluated the in vitro cytotoxicity of CsA as compared to Dexamethasone (Dex) on leukemic cells. Leukemic cells obtained from 32 B-CLL patients showed a heterogeneous pattern of spontaneous apoptosis at 24 h interval and this pattern permitted to identify: Group 1 (14/32) with high (>20%) apoptotic rate and Group 2 (18/32) with low cell death. CsA and Dex increased cell death in both groups with a different timing by an apoptotic mechanism that does not involve Bcl-2. Furthermore, in Group 2, CsA-induced apoptosis was significant higher than that observed with Dex both at 4 and 24 h. We suggest that, in B-CLL, CsA has a significant pro-apoptotic activity manifested also in patients with low spontaneous apoptosis. Our observations might be taken into account to consider new therapeutic strategies in B-CLL.

Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia-reperfusion injury in rats.

AIMS/HYPOTHESIS: A reduction in the number of endothelial progenitor cells (EPCs) is considered a plausible cause of increased cardiovascular risk in diabetes mellitus. The aim of this study was to test the hypothesis that weak bone marrow mobilisation is responsible for the decrease in circulating EPCs in diabetes. MATERIALS AND METHODS: We employed a model of hindlimb ischaemia-reperfusion (I/R) injury to study mobilisation of EPCs in control and streptozotocin diabetic rats. EPCs were defined by flow cytometry as Sca-1(+) and Sca-1(+)c-kit(+) peripheral blood cells and further characterised by the expression of CD31, von Willebrand factor and fetal liver kinase-1. Capillary density was evaluated by immunofluorescent staining of vWF. We also determined plasma levels of stromal cell-derived factor (SDF-1) and vascular endothelial growth factor (VEGF) by ELISA and muscle expression of hypoxia-induced factor (HIF-1alpha) by Western blotting. RESULTS: In control rats, EPCs showed a mobilisation curve within 7 days, while diabetic rats were completely unable to mobilise EPCs after I/R injury. As a consequence, diabetic rats showed no compensatory increase in muscle capillary density. Defective EPC mobilisation in diabetes was associated with altered release of SDF-1 and VEGF and inability to upregulate muscle HIF-1alpha. Both insulin administration and premedication with granulocyte-colony stimulating factor and stem cell factor led to partial recovery in post-ischaemic mobilisation of EPCs in diabetic rats. CONCLUSIONS/INTERPRETATION: Defective ischaemia-induced bone marrow mobilisation of EPCs impedes compensatory angiogenesis in ischaemic tissues of diabetic animals. Growth factor administration together with blood glucose control may offer a rational therapeutic strategy for diabetic ischaemic syndromes.

PDE-5 inhibitor, Vardenafil, increases circulating progenitor cells in humans.

Bone marrow-derived endothelial progenitor cells (EPCs) originate from haematopoietic stem cells in bone marrow and migrate into the peripheral circulation to promote endothelial repair and neovascularization. The number of circulating progenitor cells is reduced in patients with cardiovascular risk factor. The aim of our study was to determine the number of these cells in healthy patients and to evaluate the effect of Vardenfil, a phosphodiesterases-5 (PDE-5) inhibitor, in the number of circulating EPCs. In our study, we found a significant increase in the number of these cells after the drug administration.

Circulating endothelial progenitor cells in subjects with erectile dysfunction.

Erectile dysfunction (ED) is often the first clinical sign of endothelial dysfunction and may precede overt cardiovascular diseases. Bone marrow-derived endothelial progenitor cells migrate into the peripheral circulation to promote endothelial repair. The number of circulating progenitor cells is reduced in patients with cardiovascular risk factor. The objective of our study was to determine the number of these cells in patients with ED both with and without cardiovascular risk factors. These subjects have lower number of circulating progenitor cells, confirming the existence of an endothelial dysfunction and supplying the evidence that ED may be the first symptom of an endothelial damage.

The raft marker GM1 identifies functional subsets of granular lymphocytes in patients with CD3+ lymphoproliferative disease of granular lymphocytes.

The raft marker GM1 is expressed at very low levels at the plasma membrane of resting T cells (GM1dull). In vitro T-cell activation induces synthesis of this lipid, which is then expressed at very high levels (GM1bright) at the membrane of activated/effector cells. By flow cytometry and confocal microscopy, we analyzed the expression and organization of GM1 in a series of 15 patients with CD3+ lymphoproliferative disease of granular lymphocytes (LDGL). We found that GM1bright GL were detectable in fresh blood samples obtained in all LDGL patients, although the range of brightly stained cells was extremely variable. This distinctive in vivo pattern has never been shown in T lymphocytes from healthy individuals or in patients with different chronic T or B lymphoproliferative disorders or active infectious diseases. The low number of cycling cells detected in LDGL patients was always included within the GM1bright GL population. Interestingly, GM1bright GL were demonstrated to contain a higher amount of IFN-gamma as compared to GM1dull GL. These findings allow to distinguish subsets of GL at different levels of activation within the monoclonal CD3+ population. The GM1bright GL subset is likely to be responsible for the renewing of GL and thus for maintaining chronic proliferation.

Immortalized myeloid suppressor cells trigger apoptosis in antigen-activated T lymphocytes.

We described a generalized suppression of CTL anamnestic responses that occurred in mice bearing large tumor nodules or immunized with powerful recombinant viral immunogens. Immune suppression entirely depended on GM-CSF-driven accumulation of CD11b(+)/Gr-1(+) myeloid suppressor cells (MSC) in secondary lymphoid organs. To further investigate the nature and properties of MSC, we immortalized CD11b(+)/Gr-1(+) cells isolated from the spleens of immunosuppressed mice, using a retrovirus encoding the v-myc and v-raf oncogenes. Immortalized cells expressed monocyte/macrophage markers (CD11b, F4/80, CD86, CD11c), but they differed from previously characterized macrophage lines in their capacities to inhibit T lymphocyte activation. Two MSC lines, MSC-1 and MSC-2, were selected based upon their abilities to inhibit Ag-specific proliferative and functional CTL responses. MSC-1 line was constitutively inhibitory, while suppressive functions of MSC-2 line were stimulated by exposure to the cytokine IL-4. Both MSC lines triggered the apoptotic cascade in Ag-activated T lymphocytes by a mechanism requiring cell-cell contact. Some well-known membrane molecules involved in the activation of apoptotic pathways (e.g., TNF-related apoptosis-inducing ligand, Fas ligand, TNF-alpha) were ruled out as candidate effectors for the suppression mechanism. The immortalized myeloid lines represent a novel, useful tool to shed light on the molecules involved in the differentiation of myeloid-related suppressors as well as in the inhibitory pathway they use to control T lymphocyte activation.

Identification of a CD11b(+)/Gr-1(+)/CD31(+) myeloid progenitor capable of activating or suppressing CD8(+) T cells.

Apoptotic death of CD8(+) T cells can be induced by a population of inhibitory myeloid cells that are double positive for the CD11b and Gr-1 markers. These cells are responsible for the immunosuppression observed in pathologies as dissimilar as tumor growth and overwhelming infections, or after immunization with viruses. The appearance of a CD11b(+)/Gr-1(+) population of inhibitory macrophages (iMacs) could be attributed to high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vivo. Deletion of iMacs in vitro or in vivo reversed the depression of CD8(+) T-cell function. We isolated iMacs from the spleens of immunocompromised mice and found that these cells were positive for CD31, ER-MP20 (Ly-6C), and ER-MP58, markers characteristic of granulocyte/monocyte precursors. Importantly, although iMacs retained their inhibitory properties when cultured in vitro in standard medium, suppressive functions could be modulated by cytokine exposure. Whereas culture with the cytokine interleukin 4 (IL-4) increased iMac inhibitory activity, these cells could be differentiated into a nonadherent population of fully mature and highly activated dendritic cells when cultured in the presence of IL-4 and GM-CSF. A common CD31(+)/CD11b(+)/Gr-1(+) progenitor can thus give rise to cells capable of either activating or inhibiting the function of CD8(+) T lymphocytes, depending on the cytokine milieu that prevails during antigen-presenting cell maturation. (Blood. 2000;96:3838-3846)

Restriction of HIV type 1 infection in macrophages heterozygous for a deletion in the CC-chemokine receptor 5 gene.

Homozygosity for a 32-base pair deletion (delta32) within the CC-chemokine receptor 5 (CCR5) gene confers resistance to infection by R5-type HIV-1 isolates. To ascertain how CCR5delta32 heterozygosity influences the susceptibility of lymphocytes and macrophages to HIV-1 infection, peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDMs) from three HIV-1-uninfected CCR5delta32 heterozygous infants and three HIV-1-uninfected CCR5 wild-type homozygous infants were exposed to two R5-type primary isolates. HIV-1 infection was monitored by DNA-PCR and p24 antigen determination; CCR5 and CCR5delta32 transcripts were quantified by competitive reverse transcription-PCR. Wild-type homozygous MDMs and PBLs and heterozygous PBLs were infected by both viral isolates, albeit with different efficiencies, but heterozygous MDMs showed restriction to HIV-1 infection. Lower levels of CCR5 mRNA and protein expression were found in heterozygous versus wild-type homozygous MDMs and PBLs. Interestingly, wild-type homozygous MDMs showed higher levels of CCR5 mRNA expression compared with wild-type homozygous PBLs, while heterozygous MDMs had lower levels of CCR5 wild-type mRNA and a higher CCR5delta32/CCR5 mRNA ratio compared with heterozygous PBLs. These findings suggest that CCR5delta32 heterozygosity confers a different degree of protection against HIV-1 in PBLs and MDMs, depending on the ratio of wild-type and mutant CCR5 mRNA in the two cell types, and may delay virus spread in the host by preventing infection of monocytes and macrophages.

Unopposed production of granulocyte-macrophage colony-stimulating factor by tumors inhibits CD8+ T cell responses by dysregulating antigen-presenting cell maturation.

Tumor cells gene-modified to produce GM-CSF potently stimulate antitumor immune responses, in part, by causing the growth and differentiation of dendritic cells (DC). However, GM-CSF-modified tumor cells must be gamma-irradiated or they will grow progressively, killing the host. We observed that 23 of 75 (31%) human tumor lines and two commonly used mouse tumor lines spontaneously produced GM-CSF. In mice, chronic GM-CSF production by tumors suppressed Ag-specific CD8+ T cell responses. Interestingly, an inhibitory population of adherent CD11b(Mac-1)/Gr-1 double-positive cells caused the observed impairment of CD8+ T cell function upon direct cell-to-cell contact. The inhibitory cells were positive for some markers associated with Ag presenting cells, like F4/80, but were negative for markers associated with fully mature DC like DEC205, B7. 2, and MHC class II. We have previously reported that a similar or identical population of inhibitory "immature" APC was elicited after immunization with powerful recombinant immunogens. We show here that these inhibitory cells can be elicited by the administration of recombinant GM-CSF alone, and, furthermore, that they can be differentiated ex vivo into "mature" APC by the addition of IL-4 and GM-CSF. Thus, tumors may be able to escape from immune detection by producing "unopposed" GM-CSF, thereby disrupting the balance of cytokines needed for the maturation of fully functional DC. Further, CD11b/Gr-1 double-positive cells may function as "inhibitory" APC under the influence of GM-CSF alone.

Design, synthesis and CD4 binding studies of a fluorescent analogue of a peptide that enhances HIV-1 infectivity.

We previously demonstrated that a 23-amino-acid peptide derived from the V3 loop of the surface glycoprotein of human immunodeficiency virus (HIV-1) strain MN was able to bind soluble CD4 and to enhance HIV-1 infection. Further studies suggested that the peptide/CD4 interaction induces an increase in both CD4 expression and CD4/gp120 binding affinity. To facilitate identification of the complementary binding site for the peptide on cellular CD4, we designed an analogue carrying a single fluorescein moiety. The synthesis of this modified analogue presented several problems because of the presence of several amino acids in the sequence carrying potentially reactive groups in their side-chains, and the necessity of introducing only one marker per molecule in a position that would not affect biological activity. The side-chain of Lys19 was selected because separate studies demonstrated that its substitution with an uncharged amino acid does not reduce the peptide's biological activity. We compared the merits of various synthetic protocols used to condense the fluorescent marker with the peptide. Biological assays indicated that the presence of the fluorescein moiety did not compromise peptide binding to CD4; furthermore, binding of the labeled analogue was not abolished by trypsin treatment, suggesting that the peptide may interact with both CD4 and additional trypsin-resistant binding sites on the cell surface. Finally, we verified the preservation of HIV infection enhancing ability in the labeled peptide.

CD45 regulates apoptosis induced by extracellular adenosine triphosphate and cytotoxic T lymphocytes.

Several lines of evidence implicate protein tyrosine phosphatases (PTP) in the regulation of apoptotic cell death. We have evaluated the role of CD45, the major PTP of hematopoietic cells, in apoptosis induced by extracellular ATP (ATPe) and cytotoxic T lymphocytes (CTL). We observed that two CD45- clones obtained by mutagenesis of the Fas- cell line L1210, exhibit a higher susceptibility to apoptosis induced by ATPe, which was also evident in Ca(2+)-free conditions, when compared to the parental cell line or CD45+ variants. The CD45- cells were also more susceptible to death mediated by an alloreactive CTL clone. When the cytotoxic assay was performed in the presence of EGTA, a Ca2+ chelator, which prevents cytotoxic granule exocytosis and perforin polymerization on target cell membranes, only the CD45- target cells were killed by the CTL clone. These results suggest that a cytotoxic pathway other than the secretory or Fas-dependent pathways was responsible for the enhanced susceptibility of CD45- cells to death, and therefore provide further evidence for the role of ATPe as a possible mediator of Ca(2+)-independent target cell destruction by CTL.

c-erbB-2/neu protein expression, DNA ploidy and S phase in breast cancer.

DNA content and c-erbB2/neu protein (p185) expression were evaluated by flow-cytometry and ELISA, respectively, in 166 specimens of primary breast cancer. A non-diploid DNA content was found in 88 tumours (53%), with the DNA index ranging from 0.7-2.7. S phase fraction (SPF) evaluation, performed in 130 cases, showed significantly higher values in aneuploid than in diploid tumours (median values, 17.3% and 5.8%, respectively). Thirty-six tumours (21.6%) showed p185 overexpression, while 45 (27.1%) and 85 (51.3%) showed intermediate and low expression, respectively. A good correlation (P = 0.0023) was found between DNA content and p185 positivity. Tumours with high p185 values were mainly aneuploid, while tumours with intermediate or low expression had variable degrees of DNA content. Furthermore, p185 concentration was significantly higher in aneuploid than in diploid tumours (P = 0.009). The highest rate of p185 (+) cases and the highest p185 concentrations occurred in triploid (1.3 < D.I. < or = 1.7), compared to the other tumours. SPF values and p185 positivity rates were not significantly correlated in diploid and in aneuploid tumours.

Membrane form of TNF alpha induces both cell lysis and apoptosis in susceptible target cells.

Tumor necrosis factor alpha, in the secreted as well as membrane-associated (mTNF alpha) form, represents a cytotoxic effector mechanism of activated macrophages; in contrast, direct evidence of the mTNF alpha involvement in cytotoxic T lymphocyte (CTL)-mediated lysis has not yet been obtained. We observed that following activation with anti-CD3 monoclonal antibody (mAb), both cloned CTL and peritoneal exudate lymphocytes rapidly upregulated mTNF alpha; a similar effect was observed in the macrophage cell line J774 after stimulation with lipopolysaccharide endotoxin. Activated effector cells, which were fixed with paraformaldehyde before testing, exerted lytic activity against the TNF-sensitive WEHI 164 tumor cell line, but not against the TNF-resistant P-815 mastocytoma. This effect was completely inhibited in the presence of anti-mouse TNF alpha Ab. Moreover, both mTNF alpha-expressing macrophages and CTL induced nuclear DNA fragmentation in WEHI 164 cells, which was also blocked by anti-TNF alpha Ab and was accompanied by a morphologic degeneration characteristic of the apoptotic form of cell death. These data on the whole indicate a common mode of action for mTNF alpha expressed on different cell populations endowed with cytotoxic capability and also imply a role for this molecule in T-cell-mediated cytotoxicity.