Human cell-based anti-inflammatory effects of rosiglitazone.

PURPOSE: The C-X-C motif chemokine ligand 10 (CXCL10) participates in diabetes and diabetic cardiomyopathy development from the early stages. Rosiglitazone (RGZ) exhibits anti-inflammatory properties and can target cardiomyocytes secreting CXCL10, under interferon (IFN)γ and tumor necrosis factor (TNF)α challenge. Cardiomyocyte remodeling, CD4 + T cells and dendritic cells (DCs) significantly contribute to the inflammatory milieu underlying and promoting disease development. We aimed to study the effect of RGZ onto inflammation-induced secretion of CXCL10, IFNγ, TNFα, interleukin (IL)-6 and IL-8 by human CD4 + T and DCs, and onto IFNγ/TNFα-dependent signaling in human cardiomyocytes associated with chemokine release. METHODS: Cells maintained within an inflammatory-like microenvironment were exposed to RGZ at near therapy dose (5 µM). ELISA quantified cytokine secretion; qPCR measured mRNA expression; Western blot analyzed protein expression and activation; immunofluorescent analysis detected intracellular IFNγ/TNFα-dependent trafficking. RESULTS: In human CD4 + T cells and DCs, RGZ inhibited CXCL10 release likely with a transcriptional mechanism, and reduced TNFα only in CD4 + T cells. In human cardiomyocytes, RGZ impaired IFNγ/TNFα signal transduction, blocking the phosphorylation/nuclear translocation of signal transducer and activator of transcription 1 (Stat1) and nuclear factor-kB (NF-kB), in association with a significant decrease in CXCL10 expression, IL-6 and IL-8 release. CONCLUSION: As the combination of Th1 biomarkers like CXCL10, IL-8, IL-6 with classical cardiovascular risk factors seems to improve the accuracy in predicting T2D and coronary events, future studies might be desirable to further investigate the anti-Th1 effect of RGZ.

Management of patients with severe asthma: results from a survey among allergists and clinical immunologists of the Central Italy Inter-Regional Section of SIAAIC.

BACKGROUND: Asthma, and severe asthma in particular, is often managed within a specialized field with allergists and clinical immunologists playing a leading role. In this respect, the National Scientific Society SIAAIC (Società Italiana di Allergologia, Asma ed Immunologia Clinica), structured in Regional and Inter-Regional sections, interviewed a large number of specialists involved in the management of this respiratory disease. METHODS: A survey entitled "Management of patients with asthma and severe asthma" based on 17 questions was conducted through the SIAAIC newsletter in 2019 thanks to the collaboration between GlaxoSmithKline S.p.A. and the Inter-Regional Section of SIAAIC of Central Italy. RESULTS: Fifty-nine allergists and clinical immunologists participated to the survey, and 40 of them completed the entire questionnaire. Almost all of the specialists (88%) reported that asthma control was achieved in above 50% of their patients, even if only one third (32%) actually used validated clinical tools such as asthma control test (ACT). Poor adherence to inhaled therapy was recognized as the main cause of asthma control failure by 60% of respondents, and 2-5 min on average is dedicated to the patient inhaler technique training by two-thirds of the experts (65%). Maintenance and as-needed therapy (SMART/MART) is considered an appropriate approach in only a minority of the patients (25%) by one half of the respondents (52%). A high number of exacerbations despite the maximum inhalation therapy were recognized as highly suspicious of severe asthma. Patients eligible for biological therapies are 3-5% of the patients, and almost all the responders (95%) agreed that patients affected by severe asthma need to be managed in specialized centers with dedicated settings. Biological drugs are generally prescribed after 3-6 months from the initial access to the center, and once started, the follow-up is initially programmed monthly, and then every 3-6 months after the first year of treatment (96% of responders). After phenotyping and severity assessment, comorbidities (urticaria, chronic rhinosinusitis with or without nasal polyps, vasculitis, etc.) are the drivers of choice among the different biological drugs. In the management of severe asthma, general practitioners (GPs) should play a central role in selecting patients and referring them to specialized centers while Scientific Societies should train GPs to appropriately recognize difficult asthma and promote public disease awareness campaigns. CONCLUSIONS: This survey which collects the point of view of allergists and clinical immunologists from Central Italy highlights that asthma control is still not measured with validated instruments. There is a general consensus that severe asthma should be managed only in dedicated centers and to this aim it is essential to encourage patient selection from a primary care setting and develop disease awareness campaigns for patients.

Mesenchymal stem cells are enriched in head neck squamous cell carcinoma, correlates with tumour size and inhibit T-cell proliferation.

BACKGROUND: Cancer is a multifactorial disease not only restricted to transformed epithelium, but also involving cells of the immune system and cells of mesenchymal origin, particularly mesenchymal stem cells (MSCs). Mesenchymal stem cells contribute to blood- and lymph- neoangiogenesis, generate myofibroblasts, with pro-invasive activity and may suppress anti-tumour immunity. METHODS: In this paper, we evaluated the presence and features of MSCs isolated from human head neck squamous cell carcinoma (HNSCC). RESULTS: Fresh specimens of HNSCC showed higher proportions of CD90+ cells compared with normal tissue; these cells co-expressed CD29, CD105, and CD73, but not CD31, CD45, CD133, and human epithelial antigen similarly to bone marrow-derived MSCs (BM-MSCs). Adherent stromal cells isolated from tumour shared also differentiation potential with BM-MSCs, thus we named them as tumour-MSCs. Interestingly, tumour-MSCs showed a clear immunosuppressive activity on in vitro stimulated T lymphocytes, mainly mediated by indoelamine 2,3 dioxygenase activity, like BM-MSCs. To evaluate their possible role in tumour growth in vivo, we correlated tumour-MSC proportions with neoplasm size. Tumour-MSCs frequency directly correlated with tumour volume and inversely with the frequency of tumour-infiltrating leukocytes. CONCLUSIONS: These data support the concept that tumour-MSCs may favour tumour growth not only through their effect on stromal development, but also by inhibiting the anti-tumour immune response.

Th17 cells: new players in asthma pathogenesis.

CD4+ T effector lymphocytes are distinguished in different subsets on the basis of their patterns of cytokine secretion. Th1 cells, thank to IFN-γ production, are responsible for cell-mediated immunity against intracellular pathogens, Th2 cells, through the production of IL-4, provide some degree of protection against helminthes, and Th17 cells, via IL-17, promote neutrophils recruitment for the clearance of bacteria and fungi. However, beyond their protective role, these T-helper subsets can also be involved in the pathogenesis of several inflammatory diseases. Asthma is an inflammatory disease characterized by different clinical phenotypes. Allergic asthma is the result of an inflammatory process driven by allergen-specific Th2 lymphocytes, whereas Th17 cells are mainly involved in those forms of asthma, where neutrophils more than eosinophils, contribute to the inflammation. The identification in allergic asthma of Th17/Th2 cells, able to produce both IL-4 and IL-17, is in keeping with the observation that different clinical phenotypes can coexist in the same patient. In conclusion, a picture in which different T-cell subpopulations are active in different phase of bronchial asthma is emerging, and the wide spectrum of clinical phenotypes is probably the expression of different cellular characters playing a role in lung inflammation.

Immunomodulatory effects of BXL-01-0029, a less hypercalcemic vitamin D analogue, in human cardiomyocytes and T cells.

Current immunosuppressive protocols have reduced rejection occurrence in heart transplantation; nevertheless, management of heart transplant recipients is accompanied by major adverse effects, due to drug doses close to toxic range. In allograft rejection, characterized by T-helper 1 (Th1) cell-mediated response, the CXCL10-CXCR3 axis plays a pivotal role in triggering a self-promoting inflammatory loop. Indeed, CXCL10 intragraft production, required for initiation and development of graft failure, supports organ infiltration by Th1 cells. Thus, targeting the CXCL10-CXCR3 axis while avoiding generalized immunosuppression, may be of therapeutic significance. Based on preclinical evidence for immunoregulatory properties of vitamin D receptor agonists, we propose that a less hypercalcemic vitamin D analogue, BXL-01-0029, might have the potential to contribute to rejection management. We investigated the effect of BXL-01-0029 on CXCL10 secretion induced by proinflammatory stimuli, both in human isolated cardiomyocytes (Hfcm) and purified CD4+ T cells. Mycophenolic acid (MPA), the active agent of mycophenolate mofetil, was used for comparison. BXL-01-0029 inhibited IFNgamma and TNFalpha-induced CXCL10 secretion by Hfcm more potently than MPA, impairing cytokine synergy and pathways. BXL-01-0029 reduced also CXCL10 protein secretion and gene expression by CD4+ T cells. Furthermore, BXL-01-0029 did not exert any toxic effect onto both cell types, suggesting its possible use as a dose-reducing agent for conventional immunosuppressive drugs in clinical transplantation.

Elocalcitol inhibits inflammatory responses in human thyroid cells and T cells.

T-helper 1 (Th1) cell-mediated inflammatory responses predominate in the early pathogenesis of Graves' disease (GD), whereas Th2 cell-mediated immunity may play a role in later stages. The chemokine CXCL10 and its receptor CXCR3 are expressed in most thyroid glands of early GD patients. Circulating CXCL10 levels inversely correlate with disease duration; CXCL10 maximal expression also correlates with interferon (IFN)gamma levels in recent GD onset. Methimazole (MMI) reduces CXCL10 secretion by isolated thyrocytes, decreases serum CXCL10 levels, and promotes a transition from Th1 to Th2 dominance in patients in GD active phase. Vitamin D receptor agonists exhibit antiinflammatory properties and promote tolerance induction. We investigated the effects and the mechanism of action of a nonhypercalcemic vitamin D receptor agonist, elocalcitol (BXL-628), compared with MMI on CXCL10 secretion induced by proinflammatory cytokines. Furthermore, we studied the effects of both drugs on Th1, Th17, and Th2 cytokine secretion in CD4+ T cells. ELISA, cytometry, immunocytochemistry, Western blot, and quantitative real-time PCR were used for protein and gene analysis. In human thyrocytes, elocalcitol inhibited IFNgamma and TNFalpha-induced CXCL10 protein secretion more potently than MMI. Elocalcitol impaired both cytokine intracellular pathways, whereas MMI was effective only on the IFNgamma pathway. In CD4+ T cells, elocalcitol decreased Th1- and Th17-type cytokines, and promoted Th2-type cytokine secretion. Elocalcitol and MMI inhibited Th1 cytokine-mediated responses in thyrocytes and CD4+ T cells. In addition, elocalcitol promoted a shift toward a Th2 response. In conclusion, elocalcitol could represent a novel pharmacological tool in the treatment of autoimmune thyroid diseases.

Methimazole inhibits CXC chemokine ligand 10 secretion in human thyrocytes.

CXC chemokine ligand 10 (CXCL10) plays a pivotal role in the self-perpetuation of the inflammatory processes in patients with autoimmune thyroid disease. Treatment with methimazole (MMI) reduces serum CXCL10 in patients with Graves' disease. In isolated human thyrocytes, tumor necrosis factor (TNF)alpha demonstrates a potent synergistic effect on interferon (IFN)gamma-induced CXCL10 secretion. We investigated the mechanism underlying the synergism between IFNgamma and TNFalpha and the effect of MMI on CXCL10 secretion in human thyrocytes. A peroxisome proliferator-activated receptor gamma agonist, rosiglitazone (RGZ), a known inhibitor of T helper 1 (Th1)-mediated responses, was also studied for comparison. Experiments were carried out in human thyrocytes isolated from internodular parenchyma of thyroid tissues derived from patients who had undergone surgery for multinodular goiter. ELISA was used to measure CXCL10 levels in culture supernatant. Flow cytometry was used to assess IFNgamma membrane receptor expression. Specific mRNA analysis was performed by Taqman real-time PCR. Immunofluorescence was performed to detect nuclear translocation of nuclear factor-kappaB (NF-kappaB). In human thyrocytes, the synergistic effect of TNFalpha with IFNgamma on CXCL10 secretion is due to the upregulation of IFNgamma receptor expression. MMI decreased cytokine-induced CXCL10 secretion by reducing TNFalpha-induced upregulation of the IFNgamma receptor. RGZ decreased the cytokine-induced CXCL10 secretion by impairing NF-kappaB translocation, without affecting IFNgamma receptor. MMI and RGZ targeted thyrocytes with the same pharmacological potency, likely acting throughout different mechanisms. Targeting T helper 1-mediated autoimmune thyroid disease with drugs that impair different intracellular pathways could be a novel pharmacological tool.

Sublingual immunotherapy with Dermatophagoides monomeric allergoid down-regulates allergen-specific immunoglobulin E and increases both interferon-gamma- and interleukin-10-production.

BACKGROUND: The clinical efficacy and safety of sublingual immunotherapy (SLIT) for aeroallergens has been demonstrated in several trials, whereas the immunological changes induced by this treatment, which may account for the clinical improvement, are still unclear. OBJECTIVE: To investigate the effects of a successful SLIT on the in vitro allergen-driven T cell response and cytokine secretion as well as on the serum levels of chemokines and of IgE, IgG1 and IgG4 antibodies (Abs). MATERIALS AND METHODS: Twenty-five Dermatophagoides pteronyssinus (Dp)-sensitive patients with perennial rhinitic and/or rhinitic and asthmatic symptoms were randomized into two groups (13 untreated (UT) and 12 SLIT-treated) for a 1 year and half study. The proliferative response of peripheral blood mononuclear cell (PBMC) to purified Der p1 allergen, their cytokines (IFN-gamma, IL-4, IL-10 and TGF-beta) production and serum levels of chemokines associated with T helper type 1 (Th1) (CXCL10) or T helper type 2 (Th2) (CCL22) responses and of Dp-specific IgE, IgG1 and IgG4 Abs were evaluated before and after 6 months of treatment. RESULTS: SLIT induced a significant reduction of symptom medication scores after 6, 12 and 18 months of treatment in comparison with UT patients. SLIT-treated patients showed a significant decrease in serum levels of DP-specific IgE Abs, whereas total IgE, and specific IgG1 and IgG4 Abs remained unchanged. The proliferative response of allergen-specific T cells to Der p1 in vitro after 6 months of treatment was reduced, while no effect was observed on T cell proliferation to recall antigen (streptokinase). Moreover, Der p1-driven IFN-gamma and IL-10 were significantly increased in culture supernatants of PBMC from 6 month-treated patients in comparison with those detected at the beginning of therapy. CONCLUSIONS: These data suggest that the allergen-driven enhancement of IL-10- and IFN-gamma-producing T cells precedes and associates with SLIT-induced down-regulation of specific IgE, thus providing a rationale to explain the clinical benefit of SLIT in allergic patients.

CXCR3 and alphaEbeta7 integrin identify a subset of CD8+ mature thymocytes that share phenotypic and functional properties with CD8+ gut intraepithelial lymphocytes.

BACKGROUND: We previously demonstrated the existence of two distinct subsets of T cell receptor (TCR)alphabeta+CD8alphabeta+ single positive (SP) cells in human postnatal thymus which express the chemokine receptor CCR7 or CXCR3 and migrate in vitro in response to their specific ligands. AIM: To investigate whether these two CD8+ thymocyte subsets had distinct peripheral colonisation. METHODS: TCRalphabeta+CD8+ SP cells were obtained from normal postnatal thymus, mesenteric lymph node (LNs), small bowel, and peripheral blood (PB) specimens. Cells were then evaluated for expression of surface molecules, cytolytic potential, telomere length, and profile of cytokine production. RESULTS: CD8+CCR7+CXCR3- thymocytes exhibited CD62L, in common with those which localise to LNs. In contrast, CD8+CCR7-CXCR3+ thymocytes lacked CD62L but exhibited CD103, similar to intraepithelial lymphocytes (IELs) present in the gut mucosa where the CXCR3 ligand, CXCL10, and the CD103 ligand, E-cadherin, are highly and consistently expressed. In addition, thymocytes and gut CD8+CXCR3+CD103+ cells showed comparable telomere length, which was higher than that of PB CXCR3+CD8+ T cells. However, both of these populations contained perforin and granzyme A, and displayed the ability to produce interferon gamma and interleukin 2. Of note, CXCR3 deficient, in comparison with wild-type C57Black/6, mice showed decreased proportions of CD3+CD8alphabeta+ and increased proportions of CD3+CD8alphaalpha+ lymphocytes at gut level. Moreover, adoptive transfer of CD3+CD8alphabeta+ thymocytes from wild-type into CXCR3 deficient mice resulted in a significant increase in CD3+CD8alphabeta+ T cells in the gut mucosa but not in other tissues. CONCLUSIONS: The results of this study demonstrate the existence of a previously unrecognised subset of TCRalphabeta+CD8alphabeta+ SP CXCR3+CD103+ thymocytes which share phenotypic and functional features with CD8+ IELs, thus suggesting the possibility of their direct colonisation of the gut mucosa.

Effects of blocking urokinase receptor signaling by antisense oligonucleotides in a mouse model of experimental prostate cancer bone metastases.

An important factor implicated in tumor cell predisposition for invasion and metastasis is the malignancy-related upregulation of urokinase plasminogen activator receptor (uPAR). uPAR signals by activating different tyrosine kinases in different cells. We examined the effects of inhibiting uPAR signaling by inhibition of uPAR expression with antisense oligonucleotides (aODNs) in PC3 human prostate cancer cells and evaluated aODN effect in a mouse model of prostate cancer bone metastasis. Following uPAR aODN treatment, PC3 cells exhibited a strong decrease in uPAR expression, evaluated by flow cytometry and by polymerase chain reaction, and of FAK/JNK/Jun phosphorylation. The synthesis of cyclins A, B, D1 and D3 was inhibited, as shown by Western blotting, flow cytometry and polymerase chain reaction, and PC3 cells accumulated in the G2 phase of the cell cycle. PC3 cells' adhesion was unaffected, while proliferation and invasion of Matrigel were impaired. A total of 60 mice were subjected to intracardiac injection of PC3 cells and were randomly assigned to three groups: aODN (treated with 0.5 mg intraperitoneum/mouse/day), dODN (treated with the same amounts of a degenerated ODN) and control (injected with a saline solution). At 28 days after heart injection, mice were subjected to a digital scan of total body radiography, which revealed 80% reduction in mice affected by bone metastasis. The use of uPAR aODNs produced a substantial prophylactic effect against prostate cancer bone metastasis, which has to be ascribed to downregulation of uPAR expression.

Th1 and th2 responses, HIV-1 coreceptors, and HIV-1 infection.

The Th1/Th2 model provides an interesting paradigm for understanding several pathophysiological processes and possibly for developing new immunotherapeutical strategies. In HIV-1 infection the interaction between the type of HIV-1 strain and the pathway of the ongoing T-cell effector response, despite its complexity, may represent one of the crucial mechanisms in determining the outcome of virus infection. While the possibility of an HIV-1-driven Th1 to Th2 switch of the immune response is still debated, evidence is accumulating to suggest that cytokines produced during an immune response can contribute to promote a selective pressure toward the evolution of HIV-1 viral strains with different tropism. This article summarizes the results of our recent studies in which the expression of CCR5 and CXCR4 HIV-1 co-receptors, as well as the activity of R5- or X4- tropic strains of HIV-1 in different in vitro models of Th1/Th2 polarization was analyzed.

Reversal of human allergen-specific CRTH2+ T(H)2 cells by IL-12 or the PS-DSP30 oligodeoxynucleotide.

BACKGROUND: The chemoattractant receptor homologous molecule expressed on T(H)2 cells (CRTH2) is a receptor for prostaglandin D(2), which among human T cells is selectively expressed by T(H)2 and type 2 cytotoxic effectors. OBJECTIVE: Our purpose was to assess whether the cytokine production profile of T(H)2 effectors could be reversed by exploiting their selective expression of CRTH2. METHODS: CRTH2(+) T cells were purified from the blood of allergic subjects, stimulated with the specific allergen in the absence or presence of IL-12, and assessed by flow cytometry at the single-cell level for their ability to produce IL-4 and/or IFN-gamma after antigen or polyclonal stimulation. RESULTS: Both IL-12 and the PS-DSP30 oligodeoxynucleotide enabled CRTH2(+) allergen-stimulated T(H)2 cells to produce IFN-gamma. This change in the profile of cytokine production by T(H)2 cells from allergic subjects was related to the upregulation of IL-12 receptor beta2 chain and was associated with the loss of CRTH2. CONCLUSIONS: These data demonstrate that the cytokine production pattern of fully differentiated T(H)2 effectors can be changed to a less polarized profile, thus providing the physiologic basis for new immunotherapeutic strategies in allergic disorders.

Chemoattractant receptors expressed on type 2 T cells and their role in disease.

The existence of two functionally distinguished populations among T cells has been established in both mice and humans. Type 1 T helper (Th1) cells are involved in the defense against intracellular bacteria and many viruses, while type 2 Th cells (Th2) are the major actors in the response against parasites and play a central role in allergic inflammation. More recently, several data have suggested that some chemokine receptors are tightly regulated on T cells, and in accordance with this selective expression, Th1 and Th2 cells can be differentially recruited by specific chemokines to the inflammatory sites. Among Th2-associated chemokine receptors, CCR3, CCR4 and CCR8 have been described to play a central role in allergic inflammation. However, CCR3 is mainly expressed on basophils, eosinophils and mast cells, but it is poorly expressed by Th2 cells, and CCR4 is also expressed by Th subsets different from Th2 cells. So far, the chemoattractant receptors which among T cells appear to be selectively expressed by Th2 cells or their subsets are CCR8 and CRTH2. The ligand for CRTH2 is not a chemokine, but is prostaglandin (PG)D2, which is able to attract basophils, eosinophils, Th2 cells and type 2 cytotoxic (Tc2) CD8+ T lymphocytes. The selective expression of CRTH2 on Th2 and Tc2 cells may be useful to develop new therapeutic strategies against allergic diseases and against other immune disorders. Additional studies, however, are required to understand its effective importance in the induction and maintenance of Th2- or Tc2-mediated response and inflammation.

Chemokines and lymphopoiesis in human thymus.

Distinct and redundant chemokines are responsible for organizing the extraordinarily diverse thymocyte populations into discrete microenvironments, from the arrival of immature precursors in the thymus to the migration of different mature cell types to the periphery. We propose that, by selectively mobilizing cells, chemokines can sort positively selected cells from negatively selected cells, and that chemokines make distinctions among CD8+ subpopulations that previously have not been recognized.

Interferon-inducible protein 10, monokine induced by interferon gamma, and interferon-inducible T-cell alpha chemoattractant are produced by thymic epithelial cells and attract T-cell receptor (TCR) alphabeta+ CD8+ single-positive T cells, TCRgammadelta+ T cells, and natural killer-type cells in human thymus.

Strong reactivity for interferon-inducible protein 10 (IP-10), monokine induced by interferon gamma (Mig), and interferon-inducible T-cell alpha chemoattractant (I-TAC) was found in epithelial cells mainly localized to the medulla of postnatal human thymus. The CXC chemokine receptor common to the 3 chemokines (CXCR3) was also preferentially expressed in medullary areas of the same thymuses and appeared to be a property of 4 distinct populations: CD3+ T-cell receptor (TCR) alphabeta+ CD8+ single-positive (SP) T cells, TCRgammadelta+ T cells, natural killer (NK)-type cells, and a small subset of CD3+(low) CD4+ CD8+ TCRalphabeta+ double-positive (DP) T cells. IP-10, Mig, and I-TAC showed chemoattractant activity for TCRalphabeta+ CD8+ SP T cells, TCRgammadelta+ T cells, and NK-type cells, suggesting their role in the migration of different subsets of mature thymocytes during human thymus lymphopoiesis.

Cell cycle-dependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity.

Endothelial cell receptors for the angiostatic chemokines IFN-gamma-inducible protein of 10 kDa (IP-10) and monokine induced by IFN-gamma (Mig) have not yet been identified, and the mechanisms responsible for the effects of these chemokines on angiogenesis are still unclear. IP-10 and Mig share a common functional receptor on activated T lymphocytes, named CXC chemokine receptor 3 (CXCR3). Using in situ hybridization and immunohistochemistry, we show that CXCR3 is expressed by a small percentage of microvascular endothelial cells in several human normal and pathological tissues. Primary cultures of human microvascular endothelial cells (HMVECs) likewise express CXCR3, although this expression is limited to the S/G2-M phase of their cell cycle. Both IP-10 and Mig, as well as the IFN-gamma-inducible T-cell alpha chemoattractant (I-TAC), which all share high-affinity binding for CXCR3, block HMVEC proliferation in vitro, an effect that can be inhibited by an anti-CXCR3 antibody. These data provide definitive evidence of CXCR3 expression by HMVEC and open new avenues for therapeutic interventions in all conditions in which an angiostatic effect may be beneficial.

CRTH2 is the most reliable marker for the detection of circulating human type 2 Th and type 2 T cytotoxic cells in health and disease.

Cells expressing the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and the chemokine C receptor (CCR)4 were consistently detected in the circulation of healthy subjects, whereas numbers of cells expressing CCR3 were much lower. While all CCR4+ cells were T cells, a small proportion of CRTH2+, and about a half of the few CCR3+ cells were basophils. Only CRTH2+ T cells contained Th2 or Tc2 cells, but neither Th0 or Tc0, nor Th1 or Tc1 cells, although not all of them produced Th2-type cytokines. By contrast, CCR4+ T cells contained both Th2 or Tc2 and Th0 or Tc0 cells and even Th1 or Tc1 cells, whereas the few CCR3+ T cells were not clearly classifiable for their cytokine profile. CRTH2+ T lymphocytes were virtually devoid of chemokine CX receptor (CXCR)3+ and CCR5+ cells, but enriched in CCR3+ and CCR4+ cells. By contrast, CCR3+ or CCR4+ T cells did not show a similar clear-cut dichotomy in the expression of CCR5/CXCR3 or CCR3/ CCR4. Subjects with atopic dermatitis or HIV infection with low levels of circulating CD4+ T cells revealed a significant increase of CRTH2+ cells within both the CD4+ and the CD8+ T cell subset. These data support the concept that at present CRTH2 is the more reliable marker for detection of both human Th2 and Tc2 cells in health and disease.

Enhanced expression of the CXCR4 co-receptor in HIV-1-infected individuals correlates with the emergence of syncytia-inducing strains.

The aim of this study was to investigate the mechanisms responsible for the emergence in some HIV-1-infected individuals of highly aggressive, syncytia-inducing (SI) HIV-1 strains, which have been shown to use CXCR4 as co-receptor to enter target cells. To this end, the percentages of circulating CXCR4+CD4+ T cells were evaluated by flow cytometry in 39 untreated and 61 highly active antiretroviral therapy (HAART)-treated HIV-1-infected individuals in comparison with 35 HIV-1 seronegative subjects. Plasma viremia was also measured, and HIV primary isolates, from both untreated and HAART-treated HIV-1-infected subjects, were tested for the presence of SI strains. The results of this study showed enhanced proportions of CXCR4+CD4+ T cells in untreated patients in comparison with HAART-treated and healthy subjects. Furthermore, the results of a 12-month longitudinal study in a cohort of 11 patients undergoing HAART showed a significant reduction of CXCR4 expression after successful therapy. Finally, a significant positive correlation among the proportions of circulating CXCR4-expressing CD4+ T cells, plasma viremia, and the probability to isolate SI strains was found. These in vivo data are in keeping with previous in vitro results suggesting a bidirectional link between HIV-1 and CXCR4 expression on CD4+ T cells, and provide some clues to understanding the mechanisms exerting a selective pressure toward the emergence of SI strains.