Natural killer cell cytotoxicity in patients with recurrent herpes infections: diagnostic utility of a flow cytometric assay.

BACKGROUND: Primary immune deficiencies of natural killer (NK) cells have been described in patients with a susceptibility to herpes infections. AIMS: To assess the diagnostic utility of measurement of NK cytotoxicity in patients with recurrent oral herpes infections. METHODS: A retrospective audit was carried out on results obtained over an 18-month period, from 28 NK cell cytotoxicity assays (24 patients; all with a history of recurrent oral herpes infections), and 24 control samples (three healthy donors). Percentage specific cytotoxicity (PSC) was determined by measurement of the percentage of K562 target cells lysed by NK cells after incubation, using the NK TEST. Comparison of PSC was made with reference ranges provided. RESULTS: No patient with absent NK/NKT cells or NK cell cytotoxicity was identified (95% CI 0 to 14.8%). Two patients had persistently low PSC. Two patients with reduced PSC showed PSC within the normal reference range on repeat testing. Patient and control samples were seen both above and below the reference ranges. A relationship was expected between NK cell percentage and PSC; however this correlation was not significant (r(s)=0.29, p=0.18, 95% CI -0.14 to 0.63). CONCLUSIONS: A deficiency of NK cell cytotoxicity has not been identified in this cohort. An apparent reduction in cytotoxicity may be due to normal interpersonal and intersample variability in NK cytotoxicity. Without reference ranges established from a large population of control samples to account for this, a reduction in PSC is difficult to define. Further studies are required to identify if a correlation exists between the percentage of NK cells and PSC.

Distinctive effects of G-CSF, GM-CSF and TNFalpha on neutrophil apoptosis in systemic lupus erythematosus.

OBJECTIVE: To investigate the influence of culture with G-CSF GM-CSF and TNFalpha on neutrophil apoptosis, comparing neutrophils from SLE patients with rheumatoid arthritis (RA) patients and healthy control subjects. METHODS: Neutrophils were isolated from SLE (n= 10), RA (n= 10) and healthy control subjects (n= 10), and cultured with two different concentrations of G-CSF, GM-CSF and TNFalpha. Proportion of apoptotic neutrophils at T=0, T=2hrs and T=24hrs was measured using FITC-labelled annexinV and flow cytometry. RESULTS: Significantly more neutrophils were apoptotic at T=0 in the SLE subjects than in the other groups (median, range--Control 3.5% (0.3-7.9) SLE 9.5% (2.9-29.1) RA 3.0% (0.4-23.0) p<0.05). Following culture for 24 hours with 1ng/ml G-CSF the proportion of apoptotic neutrophils from SLE subjects was significantly increased (median, range = 51.6% (27.0-84.0) without G-CSF v 66.8% (31.8-89.2) with G-CSF p<0.05). This was not observed with RA or control subjects, in whom the trend was towards inhibition of apoptosis. Similar trends were seen with GM-CSF There was significant induction of apoptosis in SLE neutrophils after 2 hr culture with 1ng/ml TNFalpha (median, range = 2.3% (0.1-8.0) without TNFalpha v 5.2% (1.0-22.4) with TNFalpha). No significant change was seen in the other groups. There was an inverse correlation between total neutrophil count and the degree of induction of apoptosis by G-CSF and GM-CSF, determined at a range of time-points and cytokine concentrations CONCLUSIONS: Neutrophils from SLE patients display resistance to the apoptosis-inhibiting effects of G-CSF and possibly GM-CSF, and appear more susceptible to the apoptosis-inducing action of TNFalpha, the greatest resistance being observed in the more neutropenic patients.

Interleukin-10 receptor expression in systemic lupus erythematosus and rheumatoid arthritis.

OBJECTIVE: We aimed to determine the expression of the interleukin-10 receptor (IL10R) on circulating leukocytes in SLE and rheumatoid arthritis, and correlate this with plasma IL-10 levels and disease activity. METHODS: Peripheral blood was sampled from 20 SLE patients, 14 rheumatoid arthritis patients, and 14 healthy controls. IL-10R expression was determined by immunofluorescence labelling and flow cytometric analysis. Plasma IL-10 levels were measured by ELISA. RESULTS: IL-10R was highly expressed on monocytes, and to a lesser degree on neutrophils in all 3 patient groups. Only a small percentage of lymphocytes expressed IL-10R in all three groups. There was no significant difference in IL-10R expression on the surface of monocytes, neutrophils or lymphocytes in any of the 3 groups. IL-10R expression did not correlate with plasma IL-10 levels or disease activity. CONCLUSION: This study has shown no difference in surface IL-10R expression between SLE, rheumatoid arthritis and normal subjects. Deficient or excessive circulating leukocyte surface IL-10R expression therefore does not seem to play a role in the pathogenesis of SLE or rheumatoid arthritis. Functional IL-10R studies would be of interest.

Neutrophils from systemic lupus erythematosus patients demonstrate increased nuclear DNA damage.

OBJECTIVE: (i) To determine the levels of nuclear DNA damage in freshly isolated and cultured neutrophils from SLE patients (SLE), rheumatoid arthritis patients (RA) and healthy individuals and relate these to the percentage of apoptotic neutrophils. (ii) To assess rates of repair of neutrophil oxidative DNA damage. METHODS: The comet assay was used to quantify nuclear DNA damage in neutrophils from SLE patients (n = 20), control subjects (n = 15) and RA patients (n = 15). Levels of DNA damage were related to apoptosis as assessed by annexin V binding and morphology. Rates of repair of neutrophil oxidative DNA damage was measured by incorporating formamidopyrimidine-DNA glycosylase (FPG) into the comet assay. RESULTS: Nuclear DNA damage in freshly isolated and cultured (20 h) neutrophils was significantly greater in SLE patients (median = 12.5%, 27.3%; respectively) compared with RA patients (median = 9.4%, p = 0.002; 19.3%, p = 0.002; respectively) and control subjects (median = 8.2%, p = 0.003; 18.7%, p = 0.01, respectively). Significantly higher levels of circulating apoptotic neutrophils were demonstrated in SLE patients compared to RA and control subjects. Similar findings were observed following 20 h cultured neutrophil preparations. However, no significant direct correlation between neutrophil apoptosis and DNA damage was observed. Neutrophils from 3 of 5 SLE patients demonstrated an impaired ability to repair oxidatively modified DNA. CONCLUSION: Neutrophils from SLE patients display increased DNA damage and, additionally, may demonstrate defective repair of oxidative DNA damage. These features, in addition to increased rates of neutrophil apoptosis, may act as contributing factors to autoantigen excess and immune activation.

Serum and cerebrospinal fluid soluble Fas levels in clinical subgroups of multiple sclerosis.

Elevated sFas levels have been described in multiple sclerosis (MS) patients with active disease. The aim of this study was to assess the diagnostic potential of serum and cerebrospinal fluid (CSF) sFas measurements in differentiating clinically defined MS patient subgroups. Levels of sFas and sFas indices were determined in patients with stable relapsing-remitting MS (RRMS), active RRMS, primary progressive MS (PPMS), secondary progressive MS (SPMS) and patients with inflammatory (IND) and noninflammatory neurological diseases (NIND). Serum sFas modulation over 32 weeks IFN-beta1a therapy was also investigated. Serum and CSF sFas levels and sFas indices were elevated in MS compared to NIND and IND patients. Within the MS group, serum and CSF sFas levels were highest in PPMS, with active RRMS patients demonstrating the highest sFas indices. This may reflect an ongoing disease process which is occurring acutely (active disease) or incessantly (progressive disease). IFN-beta1a induced a transient increase in circulating sFas following initiation of therapy. Whilst evidence was provided for variable sFas expression in clinical subgroups of MS, there was insufficient definition between the respective groups to advocate sFas measurements as a diagnostic marker of clinical subgroups of MS.

Reduced expression of CD44 on monocytes and neutrophils in systemic lupus erythematosus: relations with apoptotic neutrophils and disease activity.

BACKGROUND: Increased numbers of apoptotic neutrophils, and impaired monocyte/macrophage clearance of apoptotic cells, have been demonstrated in systemic lupus erythematosus (SLE). CD44 is implicated in the clearance of apoptotic neutrophils. OBJECTIVE: To determine the expression of CD44 on peripheral blood monocytes and neutrophils in SLE, and examine the relations with disease activity and numbers of circulating apoptotic neutrophils. METHODS: Peripheral blood was sampled from 31 patients with SLE, 19 healthy normal subjects, and 19 patients with rheumatoid arthritis (RA). Monocyte and neutrophil density of surface CD44 expression was determined by immunofluorescence labelling and flow cytometry, and results expressed as mean channel fluorescence (MCF) values. Neutrophil apoptosis was measured by morphology in 15 patients with SLE, nine with RA, and six normal subjects. RESULTS: Monocyte CD44 expression was significantly lower in SLE (median MCF 4.71) than in healthy normal subjects (median MCF 5.61) and controls with RA (median MCF 5.39). Neutrophil CD44 expression was also significantly lower in SLE (median MCF 1.95) than in healthy normal subjects (median MCF 2.37) and controls with RA (median MCF 2.60). Monocyte, but not neutrophil, CD44 expression correlated negatively with the percentage of apoptotic neutrophils. There was no significant correlation of monocyte or neutrophil CD44 expression in SLE with disease activity or damage. CONCLUSIONS: Monocyte and neutrophil CD44 expression is reduced in SLE, and this may contribute to the impaired recognition and clearance of apoptotic neutrophils by monocyte derived macrophages.

Interferon-beta1a administration results in a transient increase of serum amyloid A protein and C-reactive protein: comparison with other markers of inflammation.

Putative markers of inflammation such as serum beta2-microglobulin and neopterin have been shown to be transiently upregulated following interferon-beta (IFN-beta) administration to multiple sclerosis (MS) patients. However, to date the role of the important inflammatory mediators serum amyloid A protein (SAA) and C-reactive protein (CRP) have not been described. Here we show that SAA but not CRP is elevated in relapsing-remitting MS patients compared to normal healthy individuals, and furthermore that both are transiently upregulated following intramuscular injection with IFN-beta1a (Avonex). This pattern of expression was found to parallel that of beta2-microglobulin and neopterin following injection and was mirrored by a selective activation of peripheral monocytes with respect to upregulation of receptors known to be involved in the inflammatory response (HLA-DR, CD16 and CD86). Injection of saline solution intramuscularly to six healthy control individuals did not produce a similar upregulation of any of the inflammatory markers investigated. Following IFN-beta1a injection, all inflammatory responses were attenuated at week 12 of therapy in comparison to those following the initial injection in a group of follow-up patients. In addition, IFN-beta1a injected on a weekly basis did not produce a sustained modulation of any of the markers investigated in patients treated for 32 weeks.

Changes in plasma cytokines induced by interferon-beta1a treatment in patients with multiple sclerosis.

It has been postulated that the efficacy of interferon-beta1a in multiple sclerosis may be due to the induction of type 2 cytokines. In this report we demonstrate that after 3 months of therapy, there is no sustained alteration in the plasma levels of type 1 (IL-12, IL-1beta and TNF-alpha) or type 2 (IL-6, IL-10) cytokines, but rather repeated induction of both with each injection. Little alteration is seen in the profile of cytokines induced with time, despite a decline in side effects. This suggests that IFN-beta1a causes repeated transient modulation of cytokine expression, but no sustained deviation in the type 1/type 2 balance.

CD80 (B7-1) and CD86 (B7-2) expression in multiple sclerosis patients: clinical subtype specific variation in peripheral monocytes and B cells and lack of modulation by high dose methylprednisolone.

Autoimmune activation of T cells by central nervous system (CNS)-derived antigens is hypothesised to underlie neural damage in multiple sclerosis (MS) patients. The role of coreceptor mediated signalling is currently under investigation in order to further elucidate the immunopathogenic mechanisms implicated and to determine possible targets for immune modulation. We have investigated whether differential coreceptor (B7-1/CD80; B7-2/CD86; CD28) expression on circulating lymphocytes and monocytes is (i) a feature of distinctive clinical subtypes of MS (relapsing-remitting in remission/stable-RRMS; relapsing-remitting in relapse/relapsing-RRMS; primary progressive/PPMS), (ii) related to disease activity, and (iii) altered by high dose corticocosteroid treatment. CD80(+) B cells were significantly reduced (P<0.05) in PPMS (4.0+/-0.8%) compared with normal subjects (CON) (9.1+/-1.1%), stable-RRMS (6.7+/-0.7%) and relapsing-RRMS (7.8+/-0.9%) patients. Comparatively fewer monocytes from relapsing-RRMS patients expressed CD86 (relapsing-RRMS 50+/-4.9% vs. stable-RRMS 75.1+/-3.4%, PPMS 77. 7+/-3.2%, CON 72.1+/-3.6%/P<0.05). Otherwise expression of coreceptors did not vary significantly between the groups. A 3-day course of methylprednisolone therapy did not alter coreceptor expression, but did suppress monocyte and B cell HLA-DR expression. There is evidence for differential coreceptor expression on circulating B cells and monocytes in MS disease subtypes. The biological significance of these findings is discussed in relation to alternative theories regarding coreceptor functioning.

Lymphocyte apoptosis in systemic lupus erythematosus: relationships with Fas expression, serum soluble Fas and disease activity.

Lupus specific autoantigens are exposed on apoptotic cells. The increased number of apoptotic lymphocytes reported in systemic lupus erythematosus (SLE) may be attributable to abnormalities of lymphocyte Fas expression or serum soluble Fas. In the present study we analysed the count of circulating apoptotic lymphocytes in SLE patients (n=50), by flow cytometry using Annexin V, compared to rheumatoid arthritis patients (RA, n=20), inflammatory bowel disease patients (IBD, n=20) and normal controls (n=20). Lymphocyte Fas expression and serum soluble Fas were measured and related to numbers of apoptotic lymphocytes. The percentage of apoptotic lymphocytes, determined by Annexin V binding, was significantly increased in peripheral blood of SLE patients (median=4.2%) compared with normal healthy donors (median=1.1%) and IBD patients (median=2. 0%) but not RA (median=3.9%). SLE lymphocyte Fas expression was not significantly different from RA or IBD patients. Serum soluble Fas in SLE patients correlated positively with apoptotic lymphocytes and antibodies to double stranded DNA. This study suggests that increased apoptotic lymphocytes and increased lymphocyte Fas expression may not be specific to SLE. Serum soluble Fas may have a role in the regulation of lymphocyte apoptosis in SLE.

Increased apoptotic peripheral blood neutrophils in systemic lupus erythematosus: relations with disease activity, antibodies to double stranded DNA, and neutropenia.

OBJECTIVE: To quantify the percentage of apoptotic peripheral blood neutrophils in systemic lupus erythematosus (SLE) and to determine the relations with disease activity and neutropenia. METHODS: Neutrophil apoptosis in SLE patients (n =50) was assessed by flow cytometry using annexin V binding and fluorescent labelled anti-fas. Rheumatoid arthritis (RA, n =20) and inflammatory bowel disease patients (IBD, n =20) were studied as disease controls. RESULTS: The percentage of apoptotic neutrophils, determined by annexin V binding, was increased in peripheral blood of SLE patients (median = 3.25%) compared with normal healthy donors (n =20, median = 1.20%) and disease controls (RA: median = 1.15%) (IBD: median = 1.15%). SLE neutrophil apoptosis correlated positively with lupus disease activity measured by SLAM score. SLE patients with increased antibodies to dsDNA (>10 mg/ml) had increased apoptotic neutrophils. Eight of 14 neutropenic SLE patients had increased apoptotic neutrophils. Increased neutrophil fas expression compared with normal controls was observed in SLE, RA, and IBD. CONCLUSION: Neutrophil fas expression is increased non-specifically in inflammatory disease. Increased circulating apoptotic neutrophils in SLE correlate positively with disease activity (SLAM) and may contribute to autoantigen excess including dsDNA.