Kinetic studies on phagocytosis IV. Cellular defects and humoral inhibition as causes of impaired neutrophil phagocytosis in sarcoidosis

Kinetic measurements of the serum-independent uptake of IgG-coated or complement-opsonized latex particles have been performed in 58 patients with sarcoidosis. The mean rate for phagocytic uptake of IgG particles was 0·56 min^-1 which was not different from that of the controls (0·59 min^-1). The phagocytosis of complement-opsonized particles was in the patient group 0·53 min^-1 and significantly (P<0·001) reduced compared to the rate of the controls (mean rate 0·94 min^-1), indicating neutrophil C3b-receptor dysfunction in sarcoidosis. PMNs from patients with sarcoidosis were not stimulated by the presence of autologous serum in contrast to PMNs from normals and in individual cases even a reduced uptake was found. More than one-third of the sarcoid sera also inhibited the phagocytosis of normal PMNs indicating the presence of a phagocytosis-inhibitory activity in sarcoid sera. Patients with more severe lung affection as estimated by measurements of total lung capacity, central airway obstruction, small airway function and pulmonary X-ray changes had a more reduced PMN phagocytosis in the presence of autologous serum than those with minor signs of lung affection (P<0·05). The phagocytosis-inhibitory activity of sarcoid serum was also more pronounced in those individuals who had high pulmonary score (P<0·05) or radiographic stage II-IV sarcoidosis (P<0·01). No correlation was found between serum levels of lactoferrin or lysozyme and any of the phagocytic variables while elevated β₂-microglobulin levels were associated with more pronounced serum-mediated inhibition of PMN phagocytosis (P<0·05). The relevance of these findings to the pathogenesis of granuloma formation in sarcoidosis is discussed.     

Corticosteroids restore the balance between locally produced Th1 and Th2 cytokines and immunoglobulin isotypes to normal in sarcoid lung

In this study, we have investigated the balance between Th1- and Th2-like activity in the lungs in sarcoidosis and have determined the effect of corticosteroid treatment on this. Twenty-one patients with acute untreated sarcoidosis were investigated by bronchoalveolar lavage (BAL) and compared with 11 normal volunteers. Sixteen of the sarcoid patients required corticosteroid therapy and seven of these were reinvestigated after 2–3 months'' treatment. In order to assess Th1- and Th2-like activity in the lungs, IgG subclasses and IgE were measured in BAL fluid and serum, and IL-2, IL-4 and interferon-gamma (IFN-γ) in BAL. In patients with untreated sarcoidosis, albumin-corrected BAL/serum ratios for IgG4 and IgE were significantly reduced (IgG4, 1.04±0.18 (mean±s.e.m.); IgE 9.58±3.11) compared with those in normal controls (IgG4 5.3±0.72, P<0.001; IgE 67.7±28.9, P<0.01). Estimates of actual levels of immunoglobulins produced in the lungs were also made and showed extremely high levels of total IgG in sarcoid patients (39.56±8.2 mg/l ) compared with controls (1.17±0.5 mg/l, P<0.001). Although there was no difference between the groups in amount of IgG4 locally produced, the proportion of total IgG which was IgG4 was greatly reduced in those with sarcoidosis (1.6±0.4% compared with 38.5±3.2%; P<0.001). Lavage levels of IL-4 were also reduced in sarcoid patients (IL-4 2.103±0.21 pg/ml) compared with those in normals (IL-4 6.8±1.05; P<0.001). Levels of IL-2 were lower (7.63±0.51 pg/ml compared with 9.4±0.95 pg/ml), but this difference was not significant. IFN-γ, however, could not be detected above 0.4 pg/ml in any of the normal lavage fluid, but was detectable in 12/21 patients with sarcoidosis (χ²=7.74; P<0.001). These changes reverted towards normal on treatment with oral corticosteroids. The mean albumin-corrected BAL/serum ratio for IgG4 before treatment was 0.88±0.33 compared with 5.5±2.1 (P<0.05) on treatment, and for IgE before treatment 9.52±2.15 compared with 50.8±17.9 (P<0.05) on treatment. Total IgG produced in the lung fell from 26.16±7.9 to 6.12±2.4 mg/l (P<0.001) on treatment, and the proportion of IgG4 locally produced rose from 2.3±0.8% to 23.9±6.1% (P<0.01). The mean level of IL-4 in lavage before treatment was 2.53±0.34 pg/ml compared with 4.7±0.34 (P<0.001) on treatment. Levels of IL-2 also rose significantly on treatment from 8.74±0.95 pg/ml before to 14.44±1.38 pg/ml (P<0.001) on treatment. Levels of IFN-γ fell from 1.65±0.43 pg/ml before treatment to undetectable levels in all patients (P<0.001) on treatment. These results demonstrate an imbalance between Th1- and Th2-like activity in the lungs in sarcoidosis, with suppression of Th2 and increase in Th1. Corticosteroid therapy restores the normal balance between Th1 and Th2 cytokines and immunoglobulins in the lungs, suggesting an effect on local immune regulation.     

Higher levels of interleukin IL‐17 and antigen‐specific IL‐17 responses in pulmonary sarcoidosis patients with Löfgren's syndrome

Sarcoidosis is a granulomatous disorder of unknown aetiology. The presence of Mycobacterium tuberculosis catalase-peroxidase (mKatG) in sarcoidosis tissue has been reported. T helper type 1 (Th1) responses against mKatG have previously been observed. However, little is known about interleukin (IL)-17 and Th17 responses in sarcoidosis. Here, we investigated the levels of IL-17 and frequencies of IL-17-producing cells responding to mKatG in sarcoidosis patients with different prognosis. Peripheral blood and bronchoalveolar lavage (BAL) cells were obtained from sarcoidosis patients with or without Löfgren''s syndrome (often associated with spontaneous recovery), and also stratified according to human leucocyte antigen (HLA) type. Cells producing IL-17 and interferon (IFN)-γ after stimulation with mKatG were enumerated by enzyme-linked immunospot (ELISPOT). The level of IL-17 in the BAL fluid of sarcoidosis patients and healthy controls was measured by quantitative immuno-polymerase chain reaction (qIPCR). We also performed flow cytometry and immunohistochemistry for further characterization of IL-17 expression. Patients with Löfgren''s syndrome had a higher frequency of IL-17-producing cells responding to mKatG in BAL fluid compared to patients without Löfgren''s syndrome (P < 0·05). The HLA-DR3⁺ sarcoidosis patients with Löfgren''s syndrome (known to have a particularly good prognosis) also had a clearly higher level of IL-17 in BAL fluid compared to healthy controls and sarcoidosis patients without Löfgren''s syndrome (P < 0·01) and (P < 0·05), respectively. No such difference between patient groups was observed with regard to IFN-γ and not with regard to either cytokine in peripheral blood. These findings suggest that IL-17-producing cells may be a useful biomarker for the prognosis of sarcoidosis and play a role in the spontaneous recovery typical of patients with Löfgren''s syndrome.     

An assessment of peripheral immunity in patients with sarcoidosis using measurements of serum vitamin D3, cytokines and soluble CD23

The aetiology of the peripheral anergy in sarcoidosis is unclear. To investigate this further we measured the serum levels of several factors important in different aspects of immune regulation to obtain a profile of those factors which promote and inhibit immune activation in sarcoidosis. Thirty-seven patients with sarcoidosis and 20 healthy controls of similar sex and age comprised the study group. Serum IL-10, interferon-gamma (IFN-γ), soluble CD23 (sCD23), IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1β and tumour necrosis factor-alpha (TNF-α) were measured using in-house ELISAs. Vitamin D3 was measured using a radioreceptor assay. Serum levels of sCD23 and IL-10 were significantly elevated in patients with sarcoidosis relative to controls (median 13.9 versus 9.5 arbitrary units/ml, P < 0.01 for sCD23, and 9.6 versus 5.0 pg/ml, P < 0.04 for IL-10). Regardless of steroid therapy or disease activity, serum levels of IFN-γ, TNF-α, IL-1β, GM-CSF and IL-8 were no different in patients with sarcoidosis and controls. Vitamin D3 levels were significantly higher in patients with sarcoidosis versus normal controls (medians 78.0 versus 56.0, P < 0.001), active sarcoidosis (n = 20) versus inactive disease (n = 17) (medians 81.5 versus 66.0, P < 0.03) and active sarcoidosis versus controls (medians 81.5 versus 56.0, P < 0.0002). The levels were no different between patients with inactive sarcoidosis and controls. We suggest that IL-10 and vitamin D3 may contribute to the peripheral anergy in sarcoidosis. The elevated serum sCD23 suggests an increase in peripheral humoral immunity. Consistent with a quiescent peripheral immune system, factors capable of monocyte/macrophage activation (TNF-α, IFN-γ, GM-CSF and IL-8) were not elevated in the peripheral circulation.     

Low ficolin-2 levels in common variable immunodeficiency patients with bronchiectasis

Common variable immunodeficiency (CVID) encompasses a heterogeneous group of antibody deficiencies characterized by susceptibility to recurrent infections and sequelae, including bronchiectasis. We investigated the relevance of the lectin complement pathway in CVID patients by analysing ficolin-2 and ficolin-3 serum levels and genotyping single nucleotide polymorphisms (SNPs) in the FCN2 and FCN3 genes. Our results show that ficolin-2 levels in CVID patients are significantly lower (P < 0·0001) than in controls. The lowest ficolin-2 levels are found in CVID patients with bronchiectasis (P = 0·0004) and autoimmunity (P = 0·04). Although serum levels of ficolin-3 were similar in CVID patients and controls, CVID patients with bronchiectasis again showed lower levels when compared to controls (P = 0·0001). Analysis of single nucleotide polymorphisms in the FCN2 gene confirmed known influences on ficolin-2 serum levels, but did not support a genetic basis for the observed ficolin-2 deficiency in CVID. We found that CVID patients with bronchiectasis have very low levels of ficolin-2. The reason for the deficiency of ficolin-2 in CVID and any possible causal relationship is currently unknown. However, as bronchiectasis is a very important factor for morbidity and mortality in CVID, ficolin-2 could also serve as biomarker for monitoring disease complications such as bronchiectasis.     

Significance of IL-1β and IL-1 receptor antagonist (IL-1Ra) in bronchoalveolar lavage fluid (BALF) in patients with diffuse panbronchiolitis (DPB)

We evaluated the effect of erythromycin therapy on pulmonary function tests and the airway inflammatory response of patients with DPB. The number of neutrophils in BALF obtained from DPB patients was significantly higher than that of healthy volunteers. Treatment with erythromycin (600 mg/day for 12·9 ± 9·5 months (mean ±s.d.)) significantly reduced the total number of cells and neutrophils in the airway, and significantly improved pulmonary function tests. The levels of IL-1β and IL-8 were significantly higher in DPB compared with healthy volunteers (P < 0·05, P < 0·05, respectively). IL-1 Ra in patients is considered to have a weak inhibitory activity for IL-1β, with approximately five-fold concentration of IL-1β compared with that in healthy volunteers (approx. nine-fold concentration of IL-1β). Erythromycin therapy significantly reduced these cytokines to levels comparable to those of healthy volunteers, and produced a trend toward reduction in the level of IL-1Ra in BALF. The level of IL-1β correlated significantly with the concentration of neutrophils in BALF (r = 0·72, P < 0·01), as well as with the level of IL-1Ra (r = 0·688, P < 0·05) and IL-8 (r = 0·653, P < 0·05). A nearly significant or significant correlation was observed between the concentration of neutrophils and levels of IL-1Ra or IL-8 in BALF (r = 0·526, P = 0·053 or r = 0·776, P < 0·01, respectively). There was also a significant relationship between FEV, and the concentration of neutrophils in BALF (r = 0·524, P < 0·05). Our results suggest that the relative amounts of IL-1β and IL-1Ra or IL-8 may contribute, at least in part, to the neutrophil-mediated chronic airway inflammation in patients with chronic airway disease, and long-term erythromycin therapy may down-regulate the vigorous cycle between the cytokine network and neutrophil accumulation, with resultant reduction of neutrophil-mediated inflammatory response.     

C1q rs292001 polymorphism and C1q antibodies in juvenile lupus and their relation to lupus nephritis

C1q deficiency is related strongly to systemic lupus erythematosus (SLE), but very few and inconsistent studies explored the single nucleotide polymorphisms of the C1q gene in relation to juvenile SLE (jSLE) and lupus nephritis (LN). The objective of this study was to analyse whether C1q rs 292001 polymorphism is associated with SLE and disease phenotype, especially nephritis, and to investigate the relation between this polymorphism and clinical data, treatment outcome, serum level of C1q protein and antibodies. Typing of C1q rs292001 polymorphism using restriction fragment length polymorphism and measuring serum levels of C1q protein and antibodies by enzyme-linked immunosorbent assay (ELISA) were performed for 130 children with SLE and 208 healthy controls. The A allele of C1q rs292001 was associated with jSLE and LN (P = 0·005 and 0·013, respectively) and the AA genotype was associated with jSLE (P = 0·036). Low serum levels of C1q protein were found in jSLE and LN (P < 0·001 and 0·009, respectively), and these levels were increased after treatment in patients with LN (P = 0·009) and active renal disease (P = 0·027). Higher titres of C1q antibodies were found in patients with LN (P = 0·015) and correlated negatively with C1q protein level (P < 0·001) and patient age (P = 0·04). The A allele and AA genotype of C1q rs292001 can be considered a susceptibility risk factor and the GG genotype could be considered protective for jSLE and LN in the studied cohort of Egyptian children. Decreased serum levels of C1q protein and increased titres of C1q antibodies may be involved in the pathogenesis of jSLE, especially LN.     

High Serum Interleukin-10 and Tumor Necrosis Factor Alpha Levels in Chronic Paracoccidioidomycosis

In patients with chronic paracoccidioidomycosis (n = 10), levels of tumor necrosis factor alpha, interleukin-10, and interleukin-2 in serum, measured by enzyme-linked immunosorbent assay (in picograms per milliliter, as mean ± standard error of the mean), were higher than in normal controls (n = 8): 186 ± 40 versus 40 ± 7 (P < 0.05), 203 ± 95 versus 20 ± 8 (P = 0.001), and 96.3 ± 78.57 versus 1.19 ± 1.19 (P = 0.045), respectively. Gamma interferon and interleukin-4 levels were similar in patients and controls.     

IL-6 and soluble IL-6 receptors (sIL-6R and sgp130) in human pleural effusions: massive IL-6 production independently of underlying diseases

IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130) levels were measured in sera and pleural effusions from 42 patients with metastatic carcinoma, non-Hodgkin's lymphoma, tuberculosis, cardiac failure and miscellaneous diseases. Pleural IL-6 levels measured by ELISA were very high in all patient groups (mean 34.8 ± 15.3 ng/ml) without significant difference according to diseases. IL-6 was shown to be biologically active in a proliferative assay. Serum IL-6 levels were low (0.049 ± 0.014 ng/ml) and did not correlate with pleural fluid levels. Pleural IL-6 levels correlated with the number of polymorphonuclear cells in pleural fluid (P< 0.03). Pleural sIL-6R levels (76 ± 8 ng/ml) were always lower than serum levels (196 ± 12 ng/ml; P< 0.0001) but correlated with them (P< 0.01). Pleural sIL-6R and albumin levels correlated (P< 0.01), suggesting a transudation of sIL-6R from the serum. Pleural sgp130 levels (10.9 ± 1.0 ng/ml) were lower than serum levels (24.6 ± 2.8 ng/ml; P< 0.002). After gel filtration of pleural fluid, the bulk of IL-6 (>90%) was recovered in a 15 000–30 000 fraction, corresponding to the expected mol. wt of free IL-6. These results suggest a production and a sequestration of IL-6 in the pleural cavity in all studied conditions.     

Increased numbers of CD5+ B cells and T cell receptor (TCR) γδ+ T cells are associated with younger age of onset in rheumatoid arthritis (RA)

Patients presenting with RA before the age of 45 years (younger onset) are known to have more aggressive disease compared with patients presenting after the age of 65 years (older onset). Coordinated expansion of circulating CD5⁺ B cell and TCR γδ⁺ T cell levels has been reported in patients with RA. This study assesses the peripheral blood levels of these two cell types in RA patients with younger and older onset of disease. CD5⁺ B cell levels were significantly elevated in the younger onset RA group (26·6 ± 4·5%) compared with the older onset RA group (14·2 ± 1·2%; P <0·01). TCR γδ⁺ T cell levels were also significantly raised in the young patients (4·0 ± 0·9%) compared with elderly patients (1·6 ± 0·2%; P <0·01). T cell levels (CD3⁺) were similar in both groups (young 66·4 ± 3·3%; old 74·3 ± 3·4% (mean ± s.e.m.); NS). Total B cell levels (CD19⁺) were also similar in these groups (7·7 ± 0·7% versus 8·9 ± 1·8%; NS). A significant positive correlation was observed between the CD5⁻ B and TCR γδ⁺ T cell types in the patients (r = 0·72, P <0.05). Compared with age-matched normal controls, the younger onset patients had similar CD5⁺ B cell and TCR γδ⁺ T cell levels to the elderly controls (CD5⁺ B cells 30·2 ± 3·0%; TCR γδ⁺ T cells 3·0 ± 0·8%). Conversely, older onset RA patients had CD5⁺ B cell levels similar to the young controls (12·3 ± 1·9%). Spontaneous in vitro synthesis of immunoglobulins (IgM, IgA and IgG) and rheumatoid factors (IgM and IgA isotypes) were not significantly different in both patient groups. The coordinate expansion of circulating CD5⁺ B cells and γδ⁺ T cells seen in patients with RA presenting before 45 years of age and not after 65 years of age may suggest a potential role for these cells in more aggressive disease states.     

Blood monocyte activation in rheumatoid arthritis: increased monocyte adhesiveness, integrin expression, and cytokine release

Infiltration of the synovium by mononuclear cells, namely lymphocytes and monocytes, is one of the main features of rheumatoid arthritis (RA) and is considered to be responsible for the development of the disease. In this study in 31 consecutive patients with RA, we investigated whether peripheral blood monocytes exhibited markers of cellular activation related to cell migration. Using flow cytometry with the respective specific antibodies, we studied the expression of integrins CD11a, CD11b, CD11c, CD49d (VLA-4), and CD49e (VLA-5) on monocytes from patients with RA and from normal (N) subjects. IL-1β, IL-6, and tumour necrosis factor-alpha (TNF-α) production by cultured monocytes was measured by immunoassay. Adhesiveness of monocytes was studied on various surfaces (plastic, human fibronectin, gelatin-coated plasma, subendothelial matrix) and on cultured endothelial cells under basal conditions or after stimulation by IL-1β. An increased number of CD14⁺ monocytes (Mo) from RA patients expressed the CD11b molecule (RA Mo = 90·3%, N Mo = 83·4%, P < 0·005). The expression of CD11b on CD14⁺ monocytes was significantly increased in RA patients (median fluorescence intensity (FI): RA Mo = 145 (range 80–466) units; normal Mo = 95 (range 24–164) units; P < 0·003). Production of extracellular IL-1β and IL-6 by RA monocytes was significantly enhanced compared with monocytes from normal subjects (IL-1β: RA = 2·65 ± 0·91 ng/ml versusN = 1·35 ± 0·85 pg/ml, P < 0·05; IL-6: RA = 4·83 ± 0·90 ng/ml versusN = 2·40 ± 0·95 ng/ml, P < 0·05). Compared with normal monocytes, RA monocytes exhibited increased adhesion to the various surfaces studied (plastic, P < 0·01; fibronectin, P < 0·01; and gelatin-coated normal or RA plasma, P < 0·01) as well as to unstimulated (P < 0·01) and IL-1β-stimulated endothelial cells (IL-1β for 4 h, P < 0·05; IL-1β for 24 h, P < 0·05). In our study, blood monocytes from RA patients exhibited features of activation related to cell adhesion.     

Calreticulin promotes angiogenesis via activating nitric oxide signalling pathway in rheumatoid arthritis

Calreticulin (CRT) is a multi-functional endoplasmic reticulum protein implicated in the pathogenesis of rheumatoid arthritis (RA). The present study was undertaken to determine whether CRT was involved in angiogenesis via the activating nitric oxide (NO) signalling pathway. We explored the profile of CRT expression in RA (including serum, synovial fluid and synovial tissue). In order to investigate the role of CRT on angiogenesis, human umbilical vein endothelial cells (HUVECs) were isolated and cultured in this study for in-vitro experiments. Our results showed a significantly higher concentration of CRT in serum (5·4 ± 2·2 ng/ml) of RA patients compared to that of osteoarthritis (OA, 3·6 ± 0·9 ng/ml, P < 0·05) and healthy controls (HC, 3·7 ± 0·6 ng/ml, P < 0·05); and significantly higher CRT in synovial fluid (5·8 ± 1·2 ng/ml) of RA versus OA (3·7 ± 0·3 ng/ml, P < 0·05). High levels of CRT are expressed in synovial membrane localized predominantly to inflammatory cells and synovial perivascular areas in both the lining and sublining layers of RA synovial tissue (RAST). Increased nitric oxide (NO) production and phosphorylation level of endothelial nitric oxide synthase (eNOS) were measured in HUVECs following CRT stimulation, while the total eNOS expression was not significantly changed. Furthermore, CRT promoted the proliferation, migration and tube formation of HUVECs, which were significantly inhibited by a specific eNOS inhibitor. These findings suggested that CRT may be involved in angiogenesis events in RA through NO signalling pathways, which may provide a potential therapeutic target in the treatment of RA.     

Usefulness of Serum Leucine-Rich Alpha-2 Glycoprotein as a Disease Activity Biomarker in Patients with Rheumatoid Arthritis

Our study aimed to investigate whether serum leucine-rich alpha-2-glycoprotein (LRG) levels are elevated in patients with rheumatoid arthritis (RA). In addition, we assessed their correlation with disease activity parameters and pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α). Our study included 69 patients with RA and 48 age- and sex-matched healthy controls. Serum concentrations of TNF-α and LRG were determined by enzyme-linked immunosorbent assay. Serum LRG concentrations were significantly elevated in patients with RA compared with those in healthy controls (30.8±14.4 vs. 22.2±6.1 ng/mL; P<0.001). In patients with RA, serum LRG levels were found to be correlated with disease activity score 28 (DAS28), erythrocyte sedimentation rate, and C-reactive protein levels (γ=0.671; γ=0.612; and γ=0.601, P<0.001, respectively), but not with serum TNF-α levels. Serum LRG levels in patients with an active disease status (DAS28≥2.6) were significantly higher than those in remission (DAS28<2.6) (36.45±14.36 vs. 24.63±8.81 ng/mL; P<0.001). Our findings suggest that serum LRG could contribute to the inflammatory process independent of TNF-α and it may be a novel biomarker for assessing inflammatory activity in patients with RA.

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Elevated Serum Homocysteine Levels Were Not Correlated with Serum Uric Acid Levels,but with Decreased Renal Function in Gouty Patients

Hyperhomocysteinemia is one of the important factors of the cardiovascular disease, and gout is well known to be associated with cardiovascular disease. There are a few reports on the serum homocysteine (Hcy) levels in patients with gout, however, the results showed discrepancies. In this study, we measured Hcy levels in patients with gout and examined factors associated with the levels of serum Hcy. Ninety-one male patients with gout and 97 age-matched healthy male controls were enrolled in the study. Serum uric acid levels were not significantly different between gout and healthy control groups. However, serum Hcy levels were significantly higher in patients with gout compared to controls (13.96±4.05 µM/L vs 12.67±3.52 µM/L, P=0.035). In gout group, patients with 1-2 stages of chronic kidney disease (CKD) had significantly lower serum Hcy than those with 3-5 stages of CKD (13.15±3.46 µM/L vs 17.45±4.68 µM/L, P<0.001). Multivariate linear analysis revealed an inverse association between serum Hcy and estimated glomerular filtration rate (eGFR) (β=-0.107, P<0.001). In conclusion, serum Hcy was elevated in male patients with gout. Hyperhomocysteinemia was not correlated with serum uric acid, but it was inversely associated with impaired renal function.

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Up-regulation of CC chemokine receptor 6 on tonsillar T cells and its induction by in vitro stimulation with α-streptococci in patients with pustulosis palmaris et plantaris

Pustulosis palmaris et plantaris (PPP) is a tonsil-related disease; tonsillectomy is somewhat effective in treating the condition. However, the aetiological association between the tonsils and PPP has not yet been elucidated fully. Recently, some chemokines and chemokine receptors, including CC chemokine receptor (CCR) 4, CCR6 and CX chemokine receptor (CXCR) 3, have been reported to play important roles in the development of psoriasis, a disease related closely to PPP. In this study, we found that CCR6 expression on both tonsillar and peripheral blood T cells was up-regulated more intensively in PPP patients than in non-PPP patients (P < 0·001 for both), but CCR4 and CXCR3 expressions were not. In vitro stimulation with α-streptococcal antigen enhanced CCR6 expression significantly on tonsillar T cells in PPP patients (P < 0·05), but this was not observed in non-PPP patients. The chemotactic response of tonsillar T cells to the CCR6 ligand CC chemokine ligand (CCL) 20 was significantly higher in PPP patients than in non-PPP patients (P < 0·05). The percentage of CCR6-positive peripheral blood T cells decreased after tonsillectomy in PPP patients (P < 0·01); this decrease correlated with an improvement of skin lesions (P < 0·05, r = −0·63). The numbers of CCR6-positive cells and the expression of CCL20 were increased significantly in pathological lesions compared with non-pathological lesions in PPP skin (P < 0·01, P < 0·05 respectively). These results suggest that a novel immune response to α-streptococci may enhance CCR6 expression on T cells in tonsils and that CCR6-positive T cells may move to peripheral blood circulation, resulting in recruitment to target skin lesions expressing CCL20 in PPP patients. This may be one of the key roles in pathogenesis of the tonsil-related disease PPP.     

An analysis of apoptosis in lymphoid organs and lupus disease in murine systemic lupus erythematosus (SLE)

Apoptosis is a programmed cell death process that helps to regulate both T cell and B cell development. In this study, we have investigated the levels of apoptotic death in cells of the thymuses and spleens (white matter) of autoimmune MRL-lpr/lpr mice with progressive lymphadenopathy and SLE disease activity; we also examined the renal pathology in these animals. Fas is a cell surface receptor, which when activated initiates the sequence of events that lead to apoptosis. In MRL-lpr/lpr mice Fas is defective, so the competency for apoptosis may be reduced. In young animals of advancing age the thymuses enlarged until in 5-month-old females the average weight was three times that at 1 month, and spleen and kidney weights also increased in size disproportionately. At light microscope level apoptotic cells in tissue sections were counted using both routine eosin and haematoxylin staining (to identify them by their morphology) and in situ end-labelling of cells with DNA strand breaks; their presence was further confirmed by electron microscopy. As the mice aged, the numbers of apoptotic cells in thymic cortex, thymic medulla and spleen white pulp areas reduced significantly (P < 0·01–0·001), whereas in BALB/c normal controls they increased significantly (P < 0·05). These changes were coincident with the development of severe lupus, whose activity was assessed by measuring serum anti-ssDNA and anti-dsDNA antibody titres and urinary protein (albumin) level which were elevated significantly by 5 months of age (P < 0·001 for both ssDNA and dsDNA and P < 0·01 for urine albumin) compared with their younger counterparts. Thus, lymphoid organ enlargement, decrease in apoptotic indices, elevated serum anti-ssDNA and anti-dsDNA antibody levels, and impaired renal function coincided with the onset and severity of lupus disease in lpr mice. It seems likely that there is a causal relationship between defective deletion of autoreactive lymphoid cells, imperfect Fas-mediated apoptosis and development of murine SLE.     

Correlations of IFN-γ-inducible protein-10 with the risk of chronic hepatitis B and the efficacy of interferon therapy in Asians

Purpose: The aim of this study was to identify the correlations of IFN-γ-inducible protein-10 (IP-10) with the risk of chronic hepatitis B (CHB) and the efficacy of interferon therapy in Asians. Method: Serum IP-10 levels were assayed using enzyme linked immunosorbent assay (ELISA) in both CHB and control group. CHB group received interferon-α2b treatment to compare the pre-treatment and post-treatment serum IP-10 levels. Relevant studies met predefined inclusion and exclusion criteria were enrolled into further meta-analysis. Stata 12.0 software was applied for data analysis. Result: Our case-control study demonstrated that CHB group had evaluated serum IP-10 levels compared with control group (285.7 ± 41.6 pg/mL vs. 79.1 ± 33.8 pg/mL, t = 21.85, P < 0.001. After treatment for 12 weeks, CHB group had remarkably decreased post-treatment serum IP-10 levels than pre-treatment (78.5 ± 20.4 pg/mL vs. 285.7 ± 41.6 pg/mL, t = 33.76, P < 0.001). No significance was observed on post-treatment serum IP-10 levels between CHB and control group (78.5 ± 20.4 pg/mL vs. 78.1 ± 33.8 pg/mL, t = 0.07, P = 0.947). Meta-analysis results demonstrated that serum IP-10 levels in CHB group were obviously higher than healthy controls (SMD = 2.21, 95% CI = 1.55~2.87, P < 0.001). A subgroup based on the HBeAg states revealed that serum IP-10 levels in both HBeAg-positive and HBeAg-negative CHB patients were notably higher than healthy controls (HBeAg-positive: SMD = 2.00, 95% CI = 1.13-2.87, P < 0.001; HBeAg-negative: SMD = 1.34, 95% CI = 0.97-1.72, P < 0.001). Conclusion: Serum IP-10 may be correlated with the risk of CHB and the efficiency of interferon therapy, thus IP-10 may be a good biomarker for the diagnosis and treatment of CHB.     

Subpopulations of human T lymphocytes. XVI. Maldistribution of T cell subsets associated with abnormal locomotion of T cells in untreated adult patients with Hodgkin's disease

Peripheral blood and splenic T cells from adult patients with Hodgkin's disease were examined for the proportions and numbers of T cells with receptors for IgM (Tμ) or IgG (Tγ) and their locomotor responses to chemotactic stimuli of casein and endotoxin-activated serum (EAS). Thirty per cent of patients had absolute lymphopenia in the peripheral blood. The proportion of Tμ cells was comparable but the proportion of Tγ cells was significantly increased (P<0·001) resulting in an abnormally low ratio of Tμ/Tγ cells when compared to those for age- and sex-matched controls. In the spleens, the proportions of T cells and Tμ cells were significantly increased (P<0·001) and Tγ cells significantly decreased (P<0·001) resulting in an abnormally high ratio of Tμ/Tγ cells when compared with normal spleens. In the peripheral blood both Tμ and Tγ cells were increased and T cells lacking either receptor (T) were significantly decreased in patients in whom spleens were involved by the tumour when compared to those in whom spleens were not involved by the tumour. Peripheral blood T cells from patients with Hodgkin's disease responded poorly to the chemotactic stimuli when compared to T cells from normal controls or T cells from the spleens of the same patients. Tμ cell proportions in patients with combined stages III and IV were significantly lower (P<0·025) than those in the peripheral blood of patients in combined stages I and II. No correlation was observed between the above parameters and histopathological types of Hodgkin's disease. This study demonstrates an abnormal distribution of T cell subsets and abnormality of locomotion of T cells between peripheral blood and spleens in patients with Hodgkin's disease. This might explain the cellular basis of at least certain immunodeficiencies so commonly associated with Hodgkin's disease.     

Correlation of disease activity and Clq-binding immune complexes with the neutrophil inclusions which form in the presence of SLE sera

Intracytoplasmic inclusions containing immunoglobulin (Ig) and complement (C3) are found in normal neutrophils (PMN) after incubation with sera from patients with SLE. These inclusions are believed to be immune complexes removed by phagocytosis from the SLE patients' sera in vitro. Similar inclusions were also noted in the circulating PMN from some patients with SLE. In the present study we have examined the relationship between the presence of intracytoplasmic inclusions and various clinical and laboratory features of SLE. Blood from forty-five patients with SLE was drawn and separated at 37°C. Fresh heparinized blood was also obtained from normal volunteers and allowed to stand for 90 min at 37°C. The buffy coat cells from both normal and SLE groups were removed, centrifuged, washed and examined (direct method) or incubated in the SLE sera for 90 min at 37°C (indirect method). Slides of washed cells were prepared in the cytocentrifuge, stained with fluorescein-conjugated goat anti-human IgG, IgM, IgA and C3 and examined under ultra-violet light.

By the direct method, 24% of patients had small intracytoplasmic inclusions in their neutrophils when stained for IgG suggesting that immune complexes were phagocytosed in vivo. None of twenty-one normal controls had similar inclusions. By the indirect method, 62% of SLE patients were positive for IgG, 15% for IgM, 8% for IgA and 31% for C3. None of the twelve normal controls were positive.

By the indirect method, PMN inclusions containing both IgG and IgM correlated with clinical activity (P<0·001), depressed serum complement (CH50, P=0·026; and C3, P<0·051), cryoglobulinaemia (P=0·014), anti-nDNA antibodies (P<0·001) and Clq-binding immune complexes (P=0·008). A suggestive correlation with granulocytopenia was also observed. The presence of inclusions containing IgG alone did not correlate with any of these parameters. C3 and IgM appeared to be mutually exclusive, i.e. neither was present simultaneously. These findings suggest (1) that normal PMN on exposure to SLE sera develop intracytoplasmic inclusions by phagocytosis of immune complexes, (2) the presence of such complexes correlates with a number of parameters of disease activity, particularly when IgG and IgM are both present and (3) such complexes may be phagocytosed in vivo as suggested by the presence of inclusions in vivo and contribute to a number of granulocyte disturbances seen in patients with SLE. These abnormalities in granulocyte function may be important, predisposing factors for infection in patients with active SLE.

     

Serum associated leucocyte locomotion inhibition and leucocyte motility in malignant melanoma: effect of BCG treatment

Serum-associated leucocyte locomotion inhibition (SALLI) and leucocyte motility were investigated in patients with malignant melanoma. Ten days after tumour excision twelve out of eighteen patients' sera exhibited a SALLI exceeding the normal range of 15%. The mean SALLI thus reached was 59·2±5·2%. No correlation was observed between SALLI and the level of invasion or the stage of the disease.

Six patients selected at random who had a mean SALLI of 71·5±5·8% after tumour excision were further treated by BCG immunotherapy and presented after 8·2±2·9 months of therapy with a significantly (P<0·01) lower SALLI of 32·6±8·1%. In eight patients treated exclusively by surgical excision, SALLI remained basically unchanged in the course of 10±2·8 months (29·0±8·0% vs 30·4±12·9%).

The mean index of leucocyte locomotion (LL) of eight melanoma patients who had received BCG for 11·2±2·3 months was 5·9±0·9 cells/field and thus significantly (P<0·01) higher in comparison with the mean index of LL (2·8±0·5) found in eight patients treated by surgical excision only 12·4±2·1 months before testing.

In addition, patients receiving BCG had a significantly higher (P<0·05) mean value of LL than fifteen healthy controls who presented with a mean index of LL of 3·4±0·3 cells/field. Our results permit the suggestion that BCG decreases SALLI and increases LL in melanoma patients.