Fluticasone induces T cell apoptosis in the bronchial wall of mild to moderate asthmatics.

BACKGROUND: Cytokines which signal via the gamma chain of the interleukin (IL)-2 receptor and the interferons (IFNs) have been shown to enhance T cell survival in vitro by rescuing cells from apoptosis. METHODS: A study was undertaken to determine whether treatment with inhaled fluticasone propionate (FP; 250 microg twice daily) for 2 weeks could modulate production of IL-15 or IFN-beta and thereby affect T cell survival in bronchial tissue of 10 patients with mild/moderate asthma. Bronchial biopsy specimens were taken before and on completion of treatment. RESULTS: The mean (95% CI) number of T cells per unit area decreased in the asthmatic group following 2 weeks of treatment with FP (from 7.0 (5.6 to 8.4) to 4.5 (4.0 to 5.1); p = 0.001). There was an increase in the percentage of T cells undergoing apoptosis following FP treatment as assessed by T cell/TUNEL staining (from 4.5 (2.6 to 6.4) to 8.7 (6.6 to 10.8); p = 0.0001). The percentage of cells staining for IL-15 and IFN-beta in the lamina propria, determined by an alkaline phosphatase biotin streptavidin technique, decreased significantly from baseline values of 31.6 (23.4 to 39.7) to 19.6 (12.5 to 26.7), p = 0.039 for IL-15 and from 18.9 (13.5 to 24.4) to 9.5 (5.9 to 13.1), p = 0.007 for IFN-beta following 2 weeks of treatment with FP. However, only the decrease in the percentage of cells staining for IL-15 was significantly correlated with an increased number of apoptotic T cells following treatment (p = 0.008). CONCLUSION: These findings support a novel mechanism for the ability of inhaled corticosteroids to decrease T cell numbers, possibly by downregulation of the cytokine IL-15.

Release of inflammatory mediators from eosinophils following a hyperosmolar stimulus.

Airway dehydration and subsequent hyperosmolarity of periciliary fluid are considered critical events in exercise-induced bronchoconstriction (EIB). It has been shown that an in vitro hyperosmolar stimulation of basophils and mast cells with mannitol can induce the release of histamine and leukotrienes. The aim of this study was to establish if a hyperosmolar challenge could trigger activation of eosinophils to release chemokines and lipid mediators. Peripheral blood eosinophils were isolated from seven asthmatic and six non-asthmatic subjects. Hyperosmolar stimulation of eosinophils with mannitol (0.7 M), resulted in a significant increase in LTC4 levels compared to baseline in both asthmatic (15.2+/-4.6 vs. 70.1+/-9.5; P = 0.0002) and control subjects (14.3+/-4.0 vs. 55.6+/-5.6; P = 0.0001). ECP levels did not increase significantly above baseline following mannitol stimulation in either group. This study shows that eosinophils can be activated by a hyperosmolar stimulus. Therefore it seems reasonable to suggest that eosinophils could contribute to EIB.

The cellular response associated with cervical intraepithelial neoplasia in HIV+ and HIV- subjects.

This study investigates local alterations in T-cell and macrophage subsets that occur in cervical epithelial neoplasia (CIN), in the presence and absence of human immunodeficiency virus (HIV) infection. Ectocervical biopsies from 10 women with CIN who were infected with HIV, and 10 women with CIN but no HIV infection were studied by immunocytochemistry. Significantly increased proportions of activated CD8+ T cells were seen in all CIN biopsies, and these proportions were further increased in the presence of HIV infection. Levels of CD8+TIA-1+ cells were particularly increased in the CIN+HIV+ group. There was a lack of expression of CD28 on the CD8+ cells of the epithelium of CIN+HIV+ samples. A significant reduction in the proportion of epithelial inductive D1+ macrophages and an increase in D1+D7+-suppressive cells were observed in the CIN+HIV+ cohort. The lack of expression of CD28 on the CD8+ cells of the epithelium of CIN+HIV+ samples in combination with the reduced CD4+ T-cell numbers seen in the presence of HIV infection may contribute to the development of higher grade CIN in this susceptible group. This may be aggravated by the reduction in the D1+ epithelial inductive macrophages, which might reflect recruitment of more suppressive D1+D7+ cells. This would further compromise the ability of the local T-cell system to respond to antigens and thus contribute to the development of neoplasia at this site. These results suggest that the increase in activated CD8+ T cells is a consequence rather than a cause of CIN.

Activated, cytotoxic CD8(+) T lymphocytes contribute to the pathology of asthma death.

This study investigates the presence of CD8(+) T lymphocytes and their possible association with viral infection in bronchi of victims of fatal asthma. Postmortem samples from the peribronchial region of the lung were obtained from seven patients who died an asthma death (AD), seven asthmatic patients who died of unrelated causes (AUC), and seven postmortem cases with no history of lung disease (control subjects). Using immunohistochemical techniques, the CD8(+) cytotoxic T-cell population in peribronchial tissue was characterized in three patient groups. The percentage of CD8(+) cells expressing the activation marker CD25 was higher in the AD group than in both the AUC and control groups (11.91 +/- 1.92% versus 3.93 +/- 1.63% and 1.09 +/- 0.56%, respectively (p < 0.001). Perforin expression, a marker of cytotoxicity, was highest in the AD group (9.16 +/- 1.5%) compared with 1.39 +/- 0.9; 1.8 +/- 0.6% in the AUC and control groups respectively (p < 0.001). Expression of interleukin-4 (IL-4) and interferon gamma (IFN-gamma) by CD8(+) T cells was higher in the AD group than the control group (p < 0.05). Furthermore, the IFN-gamma/IL-4 ratio in the AD group was less than half that of the control group (1.46 +/- 0.2 versus 3.2 +/- 0.1; p = 0.02). Using polymerase chain reaction (PCR), viral genome for rhinovirus (RV) was detected in lung tissue from three of the seven cases in the AD group. Two of these cases also had detectable respiratory syncytial virus (RSV). Viral genome for RSV was detected in five of the AUC group and in one of these cases, RV was also detected. No viral genome was detected in the lungs of the control group. In conclusion, this study provides novel evidence of an aberrant CD8(+) T-cell population, possibly in response to viral infection in subjects who die of acute asthma.

Immunity in the female lower genital tract and the impact of HIV infection.

This study investigates the distribution of immunocompetent cells in the ectocervix, and cytokine and immunoglobulin (Ig) levels in cervicovaginal secretions to determine whether they are altered in asymptomatic human immunodeficiency virus (HIV) infection. Ectocervical biopsies from 10 HIV+ and 10 presumed HIV-ve women were studied by immunocytochemistry. Levels of Igs in cervicovaginal secretions were quantified by radial immunodiffusion (RID) and cytokine levels by ELISA. HIV+ women had significantly increased numbers of CD8+ lymphocytes resulting in reversal of the CD4:CD8 ratio. There was a significant increase in the proportion of activated CD8+ HLA-DR+ and CD4+ HLA-DR + lymphocytes, but not in CD8+ TIA-1+ cells. The epithelium of the cervix from HIV+ subjects showed a significant increase in both numbers of macrophages (CD68+) and proportions of activated macrophages (CD68+ HLA-DR+) compared to normal. The stroma contained increased proportions of inductive (D1+) and suppressive (D1+ D7+) macrophages but a decrease in effector phagocyte (D7+) proportions and Langerhans' cells. Significantly lower tumour necrosis factor (TNF)-alpha levels were observed in cervicovaginal secretions from HIV+ subjects. IgG levels were 4 times higher and IgM levels twice higher in cervicovaginal secretions from HIV+ women, compared to results from normal subjects. These results suggest a response within the CD8+ cells in HIV+ women, yet these cells may have a low cytolytic capacity. The raised proportions of HLA-DR+ and D1+ CD4+ macrophages could act as antigen-presenting cells (APC) for CD4+ CD45RO+ lymphocytes, and represent a local acquired response. However, the close juxtaposition of these cells offers the potential for them to act as a local reservoir of virus and promote its proliferation. The increase of IgG over sIgA in secretions of HIV+ subjects provides evidence suggesting a dysregulation of local humoral immunity.

IFN-gamma but not IL-4 T cells of the asthmatic bronchial wall show increased incidence of apoptosis.

BACKGROUND: Previous observations have established that IFN-gamma production is depressed in CD4+ T cells from atopic asthmatics compared with non-asthmatics. OBJECTIVE: The aim of this study was to determine if decreased IFN-gamma production could be due to a dissociation between levels of apoptosis within the T cell subsets of the asthmatic bronchial wall. METHODS: Twenty asthmatics (10 atopic and 10 non-atopic) and eight non-atopic non-asthmatics underwent bronchoscopy. Cryostat sections of these biopsies were investigated using immunohistological techniques to determine the relative number of CD4/FAS+ and CD4/Bcl-2+ cells. Detection of IFN-gamma+ and IL-4+ was combined with TUNEL staining to determine the proportions of the Th1 and Th2 cells undergoing apoptosis. RESULTS: Experiments revealed raised proportions of activated CD4+ T cells as assessed by expression of HLA-DR and CD25+ expression in the asthmatic samples. Expression of Bcl-2 by the CD4+ cell population was significantly reduced in the asthmatic compared with the control group (P = 0.002). There was no significant difference in the expression of CD4+ Fas-ligand or the number of CD4+ undergoing apoptosis in the asthmatic and non-asthmatic groups. However, the IFN-gamma+ (P = 0.04) but not IL-4+ T cells in the asthmatic biopsies had significantly higher proportions of apoptotic cells compared with the control group. CONCLUSION: The evidence supports the hypothesis that Th1/Th2 imbalance in asthmatic inflammation may be a result of premature apoptosis within the Th1 subset.

Human anergic/suppressive CD4(+)CD25(+) T cells: a highly differentiated and apoptosis-prone population.

Anergic/suppressive CD4(+)CD25(+) T cells exist in animal models but their presence has not yet been demonstrated in humans. We have identified and characterized a human CD4(+)CD25(+) T cell subset, which constitutes 7-10 % of CD4(+) T cells in peripheral blood and tonsil. These cells are a CD45RO(+)CD45RB(low) highly differentiated primed T cell population that is anergic to stimulation. Depletion of this small subset from CD4(+) T cells significantly enhances proliferation by threefold in the remaining CD4(+)CD25(-) T cells, while the addition of isolated CD4(+)CD25(+) T cells to CD4(+)CD25(-) T cells significantly inhibits proliferative activity. Blocking experiments suggest that suppression is not mediated via IL-4, IL-10 or TGF-beta and is cell-contact dependent. Isolated CD4(+)CD25(+) T cells are susceptible to apoptosis that is associated with low Bcl-2 expression, but this death can be prevented by IL-2 or fibroblast-secreted IFN-beta. However, the anergic/suppressive state of these cells is maintained after cytokine rescue. These human regulatory cells are therefore a naturally occurring, highly suppressive, apoptosis-prone population which are at a late stage of differentiation. Further studies into their role in normal and pathological situations in humans are clearly essential.

Macrophages : identification, separation, and function.

Within the human lung, macrophages can be found in the pleura, interstitium, alveoli, airways, vasculature, and walls of the bronchi and bronchiols. This distribution does not simply reflect the ubiquitous nature of these cells, as the macrophages found at these different sites show subtle distinctions in terms of cell physiology and phenotype (1). Further, animal studies have revealed functional differences between macrophages from different lung compartments (2). These differences may however be more apparent than real. Macrophages are motile cells and those, for example, present in the airways may arrive via the lung interstitium and are known to be capable of migrating back into the tissues of the lung. Thus, any observed differences between cells in different compartments are likely to be a reflection of the particular environment the cells find themselves in, rather than definitive distinctions between cell types (reviewed in 3). The message is that macrophages are "plastic" in terms of their phenotype. As different phenotypes have been shown to reflect different functions, it would seem inevitable, therefore, that these cells also exhibit a diversity of function. Indeed, it is now recognized that the phagocytic scavenger or microbicidal effector cell, are just two of several roles these cells can play.

Designing bronchial biopsy studies.

Bronchial biopsy provides valuable information about the inflammatory processes in lung tissue, but optimal results are only achieved if the design of intervention studies is sufficiently rigorous. The parallel-group design has merit, but the cross-over design is statistically superior, providing the wash-out period is effective. Heterogeneity of contributing pathologies in asthma patients results in large inter-patient variability which must be controlled for, for example by using strict inclusion criteria, which should ideally relate to the specific inflammatory marker being studied. The inclusion of a placebo group helps to quantify sample variability. The study must have sufficient statistical power to detect inter-group differences for each variable; appropriate adjustments should be made when multiple tests are used. Studies with larger patient numbers are best performed using a multi-centre design, with one centre analysing all tissue samples to reduce variability. Study duration depends on the type of investigation, but should ideally be short. Longer studies are necessary to evaluate chronic changes such as tissue remodelling. Changes in clinical status and cellular events may follow different time courses after intervention. Biopsy measurements are less reproducible than physiological tests, and diurnal variation in the number and function of inflammatory cells can further complicate measurement. The timing of clinical trial assessments needs to allow for these idiosyncrasies. Finally, a balance must be maintained between the risk, albeit small, and the benefit of performing bronchial biopsies.

The anti-inflammatory profile of inhaled corticosteroids: biopsy studies in asthmatic patients.

The beneficial effects of inhaled corticosteroids in the treatment of asthma are well established. A potent topical anti-inflammatory action is assumed to underlie the therapeutic effect, given that these agents alter the number and function of a range of inflammatory cells and markers in airway biopsies. This activity profile is shown by all inhaled corticosteroids, in a variety of patient types and study designs. Thus, treatment with inhaled corticosteroids leads to consistent reductions in the number and activation of mast cells and eosinophils in biopsy specimens. Other relevant findings include reductions in T-lymphocytes, which contribute to chronic inflammation via the secretion of pro-inflammatory cytokines (some of which are responsible for eosinophil accumulation and activation). Inhaled corticosteroids may therefore act by down-regulating immunoreactivity, so reducing activation of T lymphocytes and (consequently) eosinophils. There is considerable interest in whether corticosteroids can inhibit or reverse some structural changes in the airways, including basement membrane thickening, collagen deposition and increased airway vascularity; it has been suggested that these changes may contribute towards airway hyperresponsiveness and irreversible airway obstruction. In summary, inhaled corticosteroids have a broad spectrum of anti-inflammatory activity in asthma patients, but the relationship between changes in clinical and immunopathological parameters, particularly in the long-term, requires further study.

The inhibition of cutaneous T cell apoptosis may prevent resolution of inflammation in atopic eczema.

Atopic eczema (AE) is characterized by the persistence of infiltrating T lymphocytes in the dermis. To test the hypothesis that dysregulation of normal T cell apoptosis may contribute to the pathogenesis and chronicity of AE we compared patients with a normal resolving immune response (Mantoux reaction (MR)) induced in healthy volunteers by cutaneous PPD injection. Significantly less T cell apoptosis was observed in lesional skin of AE patients compared with either the peak or the resolution phase of the MR (P < 0.0001). The low incidence of T cell apoptosis in AE was associated with significantly increased levels of Bcl-2 relative to Bax (P < 0.0001) and significantly decreased CD95-L expression (P < 0.002) compared with the resolving MR. The cytokines IL-15 and interferon-beta (IFN-beta), which prevent activated T cell apoptosis, were expressed maximally on day 7 and day 14 of the MR, respectively. In contrast, AE patients expressed high levels of both IL-15 and IFN-beta in cutaneous lesions at the same time. This suggests that the co-expression of two anti-apoptotic cytokines, which are not found together during resolving cutaneous responses, may contribute to excessive T cell survival which leads to the persistence of inflammation in patients with AE.

Macrophage subpopulations and macrophage-derived cytokines in sputum of atopic and nonatopic asthmatic subjects and atopic and normal control subjects.

BACKGROUND: Previous studies have shown a prominent macrophage signal in the bronchial mucosa from nonatopic (intrinsic) compared with atopic (extrinsic) asthmatic subjects. This observation might have represented an expansion of a proinflammatory macrophage population or a homeostatic mechanism to decrease T(H)2-type inflammation. OBJECTIVE: The aim of the study was to investigate the numbers of macrophages and macrophage subpopulations and the expression of IL-10 and IL-12 in sputum from asthmatic and control subjects. METHODS: Eight atopic asthmatic (AA) subjects, 10 nonatopic asthmatic (NAA) subjects, 6 atopic control (AC) subjects, and 7 normal control (NC) subjects underwent sputum induction. Macrophages were enumerated by using Romanowsky stain and immunocytochemistry (CD68). RFD1 (interdigitating cell marker) and RFD7 (mature phagocyte marker) mAbs were used for immunocytochemical phenotyping, whereas IL-10 and IL-12 messenger (m)RNA was examined with in situ hybridization by using (35)S-labeled riboprobes. The phenotype of cells expressing IL-10 or IL-12 mRNA was examined by simultaneous in situ hybridization and immunostaining. RESULTS: No differences in the numbers of CD68(+) macrophages and RFD1(+), RFD7(+), and RFD1(+)/RFD7(+) subpopulations were found between AA, NAA, AC, and NC subjects. However, the numbers of IL-10 and IL-12 mRNA(+) cells were increased in AA subjects compared with NAA, AC, and NC subjects (P <.05). No other differences were found among the groups. Most of the IL-10 and IL-12 mRNA(+) cells in sputum from asthmatic subjects were macrophages (>80%), with less than 10% of mRNA colocalizing to epithelial cells. CONCLUSIONS: Sputum macrophage numbers, unlike tissue macrophages, as previously reported, were not elevated in NAA subjects. Increased IL-10 and IL-12 expression in atopic asthma may indicate the existence of a homeostatic mechanism to decrease lung inflammation. The lack of such cytokines in intrinsic asthma may predispose to bronchial inflammation in these subjects.

Fluticasone propionate-induced regulation of the balance within macrophage subpopulations.

In asthma, treatment with inhaled corticosteroids reduces chronic peribronchial inflammation and restores the balance within macrophage subpopulations. This study investigates whether corticosteroids can regulate monocyte differentiation in vitro and thereby influence the balance of functionally distinct macrophages. Graded doses of fluticasone propionate (FP) were added to cultures of normal peripheral blood monocytes in the presence or absence of IL-4. Cells were harvested after 7 days' culture. Double immunofluorescence studies were performed on cytospins of differentiated macrophages using the MoAbs RFD1 and RFD7 to distinguish inductive and suppressive macrophages by their respective phenotypes. Macrophage function was determined by quantifying allostimulation in a mixed leucocyte reaction and by measuring tumour necrosis factor-alpha (TNF-alpha) production. FP reduced the number of mature cells with a D1+ antigen-presenting phenotype and up-regulated the development of cells with the D1/D7+ and D7+ phenotypes. Functionally, this was associated with reduced stimulation of T cell proliferation in a mixed leucocyte reaction (MLR). Fluticasone also reversed the increase in both D1+ expression and TNF-alpha production induced by IL-4. The effect of FP persisted for 24 h after removal of FP from the culture medium. These results suggest that FP treatment of asthmatics may have a direct beneficial effect by normalizing the macrophage subset imbalance that contributes to the chronic peribronchial inflammation present in this condition.

The reproducibility of repeat measures of airway inflammation in stable atopic asthma.

Measures of airway inflammation are increasingly being used as outcome measures in asthma intervention studies. Meaningful interpretation of observed changes in bronchial mucosal cell numbers should depend, in part, on the reproducibility of repeat measures over time. We wanted to investigate the reproducibility of immunopathologic and physiologic parameters after short and long measurement intervals. We therefore performed spirometry, bronchial provocation challenge, and fiberoptic bronchoscopy with endobronchial biopsy (always right upper lobe second-generation bronchus) at baseline, after 2 wk, and again after 8 wk on nine subjects with stable atopic asthma (receiving inhaled placebo and beta-agonist therapy only). Numbers of T cells, memory T cells (CD45Ro(+)), macrophages (CD68(+)), and eosinophils (EG1(+) and EG2(+)) on immunohistochemical stains of bronchial biopsies were quantified by computerized image analysis. Intraclass correlation coefficients (ICCs) of reproducibility were calculated for repeat measures of each parameter and a high ICC (greater than 0.6) was interpreted as "highly reproducible." Repeat measures of FEV(1), FEF(25-75%), and PC(20) were highly reproducible after short (2-wk) and long (8-wk) intervals. Only repeat measures of EG2(+) had an ICC greater than 0.6 after 8 wk. Repeat measures of CD45Ro(+), EG2(+), and T cell numbers (but not CD68(+) and EG1(+) cells) are highly reproducible and reliable parameters of asthmatic airway inflammation after a 2 wk interval.

Phenotypic analysis of IL-10-treated macrophages using the monoclonal antibodies RFD1 and RFD7.

Suppressive or tolerogenic antigen-presenting cells (APC) might play an important role in the control of auto/hyperreactivity and the resolution of the immune response. Recent studies have provided evidence that tolerogenic APC can be induced by anergic T cells or interleukin-10 (IL-10). The aim of this study is to investigate how anergic T cells and IL-10 induce the suppressive APC phenotype and how this affects the immune response. Previously, two monoclonal antibodies (RFD1 and RFD7) were described by our lab which distinguish inductive (RFD1+RFD7-), phagocytic (RFD1-RFD7+) and suppressive (RFD1+RFD7+) macrophages. RFD1 recognizes an MHC class II-associated epitope which has restricted expression, and RFD7 recognizes a predominantly cytoplasmic antigen. Macrophages were derived from the adherent fraction of peripheral blood mononuclear cells from healthy donors. At day 5, IL-10 or IFNgamma (a cytokine which should lead to the inductive APC phenotype) was added to the cultures. At day 7, the macrophages were harvested and their phenotypes were assessed by immunohistochemical staining and FACS analysis. Upon culture of macrophages with IL-10 RFD1 staining and HLA class II expression were reduced, whereas RFD7 staining was increased. Incubation of APC with IFNgamma led to upregulation of RFD1 and HLA class II, without affecting RFD7 staining. This suggests that IL-10 induced the suppressive RFD1+RFD7+ APC population, whereas IFNgamma treatment led to the inductive RFD1+RFD7- APC subset. Thus the use of IL-10 and/or IFNgamma, and the discrimination offered by mAbs RFD7 and RFD1 represent a model whereby APC function in terms of T cell stimulation or T cell anergy can be assessed.

Dysregulation of monocyte differentiation in asthmatic subjects is reversed by IL-10.

BACKGROUND: IL-10 can modulate the differentiation of normal monocytes to macrophages, increasing the proportion of maturing cells with a phenotype consistent with T cell suppressive activity. Analysis of the immunopathology in endobronchial biopsies from asthmatic subjects has revealed significantly reduced proportions of suppressive macrophage populations associated with chronic T-cell mediated inflammation. OBJECTIVE: This study investigates whether the altered homeostasis within the lung macrophage populations in asthma is reflected in aberrant differentiation of peripheral blood monocytes and whether this differentiation may be influenced by IL-10. METHODS: Monocytes from 14 normal individuals and 14 atopic asthmatics were grown in culture for 7 days in the presence or absence of IL-10, added on day 5. Double immunofluoresence studies were performed on cytospins of the differentiated macrophages using the monoclonal antibodies RFD1 and RFD7 to distinguish inductive and suppressive macrophages by their respective phenotypes. HLADR expression was quantified using the monoclonal antibody RFDR1. Macrophage function was determined by quantifying allostimulation in a mixed leucocyte reaction and by measuring TNFalpha and TGFbeta production. RESULTS: With no cytokine addition the proportion of maturing macrophages with a suppressive phenotype (D1+D7+) at day 7 was lower in the asthmatic samples (18%) compared with normals (25%). IL-10 increased the proportion of suppressive cells in cultures of both asthmatic and normal monocytes with the increase in the asthmatic subjects (94% increase) being significantly greater than that in normal subjects (32% increase) (P<0.01). Asthmatic monocytes had a greater effect in stimulating MLR than normals (P < 0.05) but the addition of IL-10 reduced T cell proliferation in an MLR to a equivalent level in both groups. CONCLUSIONS: These results suggest that a fundamental problem may exist in the differentiation of monocytes in asthma which may be reversed by IL-10.

The role of apoptosis in the resolution of T cell-mediated cutaneous inflammation.

We have investigated cutaneous purified protein derivative-induced delayed-type hypersensitivity (DTH) responses in healthy volunteers to determine features associated with both the generation and resolution of the reaction. The clinical peak of the response occurred at day 3; however, T cell numbers were maximal on day 7. There was a preferential increase of CD4+ CD45RO+ T cells on day 7, which was largely due to proliferation, since a mean of 19% was in cycle. The proliferation of this subset was associated with the presence of IL-15, which was expressed as early as 12 h, and IL-2, which showed peak expression at 7 days. By day 14, there was a significant decrease in both the mean T cell number/unit area and IL-2 and IL-15 expression in perivascular infiltrates. Maximal CD95 (Fas/Apo-1) ligand and TNF-alpha expression were observed at 7 days and were associated with the presence of 1.83% (range 0.81-2.48%) apoptotic T cells. At 14 days, CD95 ligand and TNF-alpha expression were reduced significantly, and the presence of 2.5% (range 1.5-3.75%) of apoptotic T cells at this time was probably due to cytokine deprivation, associated with decreased Bcl-2 relative to Bax expression. The induction and resolution of the Mantoux reaction may depend on the expression of cytokines, such as IL-2 and IL-15, which regulate both proliferation and apoptosis in T cells. Failure to control either of these phases of the Mantoux reaction may contribute to the chronicity of inflammatory responses in certain cutaneous diseases.

Changes to the cytokine microenvironment in the genital tract mucosa of HIV+ women.

As previous studies have indicated that genital tract mucosal T cell function may be impaired in HIV infection, we investigated the T cell cytokine mRNA in the genital tract mucosa of HIV-infected women to determine if there are alterations in the cytokine profile which may explain the T cell impairment. The in situ hybridization technique was used to investigate the T helper-1 (Th1: IL-2, interferon-gamma (IFN-gamma)) and Th2 cytokine (IL-4, IL-5, IL-10) mRNA profile in cervical biopsies from 10 HIV+ and 10 HIV- subjects. Cervical intraepithelial neoplasia (CIN) and genital infection had previously been excluded and the distribution of immunocompetent cells within the cervical mucosa was known for each subject. Non-parametric tests were used to compare the optical density (OD) of cytokine mRNA in the HIV+ and HIV- groups. Comparisons were also made between peripheral CD4 lymphocyte counts, cervical CD4/CD8 T lymphocyte ratios and cytokine mRNA OD in HIV+ subjects. The HIV+ women had significantly higher mRNA OD for the Th2 cytokines IL-4, IL-5 and IL-10 than HIV women. There was also significantly lower IL-2 mRNA OD in the former group. HIV+ women had lower IFN-gamma mRNA than HIV- women, but the difference was not statistically significant. There was no correlation between cytokine mRNA OD and peripheral CD4 count or cervical CD4/CD8 ratio. The predominance of Th2 cytokines, which are immuno-inhibitory, in the cervical mucosa of HIV+ women may underlie the impaired cytotoxic potential observed in the CD8+ T lymphocytes and may contribute to the susceptibility of HIV-infected women to recurrent genital tract infections and cervical neoplasia.

Changes in lung lymphocyte populations reflect those seen in peripheral blood in HIV-1 positive individuals.

We have investigated the level of lymphocytosis present in the lung of human immunodeficiency virus (HIV)-1+ infected patients with and without pulmonary disease and how changes in natural killer (NK), B and T-cells seen in peripheral blood (PB) compare with those seen in bronchoalveolar lavage fluid (BALF). Lymphocyte subpopulations and their expression of activation, cytotoxic markers and memory status were characterized by triple immunofluorescence. Macrophages accounted for over 80% of the BAL cells. Only three out of 72 patients had a lymphocyte percentage >30%. No statistically significant differences in the relative proportions of NK, CD4 and CD8 populations were seen in BALF when compared to PB, except for a twofold increase in the percentage of activated CD8 cells in BALF. The only differences in BALF populations between the HIV-1+ groups were a lower percentage of CD4+ cells, and a higher percentage of activated CD8+ cells in the patients with pneumonitis. In the present cohort of patients there was little evidence for an overall lymphocytosis in bronchoalveolar lavage fluid of HIV-1+ subjects. Changes observed in lymphocyte subsets of bronchoalveolar lavage fluid populations reflected those in peripheral blood, and were similar for patients with and without pneumonitis. Evidence of increased CD8 subset activity in bronchoalveolar lavage fluid did, however, emerge.