Theo A. Out

Selective accumulation of differentiated CD8+T cells specific for respiratory viruses in the human lung

The lungs are frequently challenged by viruses, and resident CD8+ T cells likely contribute to the surveillance of these pathogens. To obtain insight into local T cell immunity to respiratory viruses in humans, we determined the specificity, phenotype, and function of lung-residing CD8+ T cells and peripheral blood CD8+ T cells in a paired analysis. The lung contained markedly higher frequencies of influenza (FLU)-specific and respiratory syncytial virus (RSV)-specific CD8+ T cells when compared with the circulation. This contrasted with an equal distribution of cytomegalovirus- and Epstein-Bar virus–specific CD8+ T cells. Noticeably, a substantial fraction of the lung-residing FLU- and RSV-specific CD8+ T cells had progressed to a relatively late differentiation phenotype, reflected by low expression of CD28 and CD27. Lung-derived FLU-specific CD8+ T cells had low activation requirements, as expansion of these cells could be initiated by cognate peptide in the absence of helper cell–derived signals. Thus, the human lung contains high numbers of differentiated FLU- and RSV-specific memory CD8+ T cells that can readily expand upon reexposure to virus. Resident lung T cells may provide immediate immunological protection against pulmonary virus infections.

Interleukin-8 in airway inflammation in patients with asthma and chronic obstructive pulmonary disease

We have investigated whether IL-8 is present in airway secretions from patients with asthma and chronic obstructive pulmonary disease (COPD) to obtain information on its possible role in airway inflammation in obstructive airways disease. In the bronchoalveolar lavage fluid (BALF) from 11 clinically stable patients with asthma the levels of IL-8 were increased compared to 10 healthy subjects (median: controls 21.5 pg/ml, asthma 244 pg/ml: p < 0.005). In the patients with asthma the levels of IL-8 correlated with the percentage neutrophils in the BALF (r = 0.81; p < 0.001) and with a parameter of the permeability of the respiratory membrane, the quotient (alpha 2-macroglobulin in BALF)/(alpha 2-macroglobulin in serum) (r = 0.66; p < 0.025). In the sputum sol phase of 9 patients with symptomatic asthma the levels of IL-8 were lower than in 9 patients with COPD (asthma: 6.4 ng/ml; COPD: 16.3 ng/ml; p < 0.02) and significantly correlated with those of neutrophilic myeloperoxidase (MPO; r = 0.85; p < 0.005). The increased levels of IL-8 in the airway secretions from both patients with asthma and COPD may be markers of an ongoing inflammatory process, which is more pronounced in patients with COPD. In patients with asthma the strong correlation between the levels of IL-8 and the percentage neutrophils and/or the levels of MPO points to a role of IL-8 in the recruitment and activation of neutrophils in the airway lumen.

B lymphocyte autoimmunity in rheumatoid synovitis is independent of ectopic lymphoid neogenesis

B lymphocyte autoimmunity plays a crucial role in the pathogenesis of rheumatoid arthritis. The local production of autoantibodies and the presence of ectopic lymphoid neogenesis in the rheumatoid synovium suggest that these dedicated microenvironments resembling canonical lymphoid follicles may regulate the initiation and maturation of B cell autoimmunity. In this study, we assessed experimentally the relevance of ectopic lymphoid neogenesis for B cell autoimmunity by a detailed structural, molecular, and serological analysis of seropositive and seronegative human synovitis. We demonstrate that synovial lymphoid neogenesis is a reversible process associated with inflammation which is neither restricted to nor preferentially associated with autoantibody positive rheumatic conditions. Despite the abundant expression of key chemokines and cytokines required for full differentiation toward germinal center reactions, synovial lymphoid neogenesis in rheumatoid arthritis only occasionally progresses toward fully differentiated follicles. In agreement with that observation, we could not detect Ag-driven clonal expansion and affinity maturation of B lymphocytes. Furthermore, ectopic lymphoid neogenesis is not directly associated with local production of anti-citrullinated protein Abs and rheumatoid factor in the rheumatoid joint. Therefore, we conclude that synovial lymphoid neogenesis is not a major determinant of these rheumatoid arthritis-specific autoantibody responses.

Respiratory syncytial virus-specific CD8+ memory T cell responses in elderly persons.

BACKGROUND We investigated respiratory syncytial virus (RSV)-specific CD8(+) memory T cell responses in healthy control participants (n=31) and in patients with chronic obstructive pulmonary disease (COPD) (n=9), with respect to frequency, memory phenotype, and proliferative requirements. METHODS The properties of RSV-specific CD8(+) T cells were analyzed by use of RSV tetramers. The proliferative requirements of RSV-specific CD8(+) T cells were analyzed by culture of peripheral-blood mononuclear cells with RSV peptide in combination with distinct cytokines. RESULTS RSV-specific CD8(+) memory T cells showed a high level of expression of CD27 and interleukin-7R alpha and a low level of expression of CCR7. In the healthy participants, the frequency of RSV tetramer(+) CD8(+) T cells was significantly lower than the frequency of influenza virus A (FLU) tetramer(+) CD8(+) T cells (P=.0001). In contrast to FLU tetramer(+) CD8(+) T cells, we could detect RSV tetramer(+) CD8(+) T cells in the subgroup of elderly healthy participants (age, > or =55 years) and in the patients with COPD only after in vitro expansion. Expanded RSV-specific T cells produced interferon- gamma and granzyme B. CONCLUSION We provide evidence that a pool of functional RSV-specific CD8(+) memory T cells persists in the peripheral blood of healthy individuals and patients with COPD. Low numbers of RSV-specific memory T cells in the elderly and in patients with COPD may explain the increased susceptibility to RSV infection in these populations.

Immune Responses and Prediction of Major Infection in Patients Undergoing Transhiatal or Transthoracic Esophagectomy for Cancer

ObjectiveTo investigate alterations in immune responses after transhiatal versus transthoracic esophageal resection and to evaluate the role of preoperative immune functions in predicting postoperative infectious complications. Summary Background DataImpaired immune defense is associated with a decreased resistance to infection. Patients undergoing esophageal resection via a transhiatal or transthoracic approach are prone to develop infectious complications. There are no randomized data on immune responses after two major surgical interventions. MethodsThe study group consisted of 20 patients who were randomly allocated to a limited transhiatal or extended transthoracic esophagectomy for cancer. Blood samples were taken before the operation and at regular intervals thereafter from day 1 to day 10. Monocyte and T-helper type 1 (Th1) and type 2 (Th2) lymphocyte functions were assessed in stimulated whole blood cultures. ResultsBoth surgical groups had severely depressed in vitro production of interleukin (IL)-12, IL-10, interferon-&ggr;, IL-2, IL-4, and IL-13 on postoperative day 1. Depression of Th2-type cytokine production was more profound after transthoracic than after transhiatal esophagectomy (IL-4, P = .005; IL-13, P = .007). Postoperative reduction in Th1-type cytokine production was similar between the two groups (interferon-&ggr;, P = .40; IL-2, P = .06). Irrespective of the surgical approach, patients who developed major infectious complications after surgery presented with a diminished T-cell cytokine production before the operation compared to those who had a relatively uneventful recovery (IL-4, P = .045; interferon-&ggr;, P = .064). In regression analysis, the occurrence of postoperative major infection was best predicted by increased duration of anesthesia (P < .0001) and low preoperative interferon-&ggr; production (P = .006). ConclusionsBoth transhiatal and transthoracic esophagectomy induced severely depressed monocyte and T-lymphocyte cytokine production. The extent of the surgical procedure had a differential immunosuppressive impact on Th2-type but not on Th1-type cell activity, indicating that the two Th pathways were downregulated through distinct mechanisms. Preoperative interferon-&ggr; determination would be useful to anticipate the occurrence of postoperative major infectious complications.

The value of rheumatoid factor and anti-citrullinated protein antibodies as predictors of response to infliximab in rheumatoid arthritis: an exploratory study

OBJECTIVE It remains unclear whether autoantibodies are useful biomarkers to tailor the choice of biological treatment in RA. We investigated the relationship between the presence and levels of different RF and ACPA isotypes and the response to TNF blockade in an exploratory study. METHODS A total of 101 active RA patients were prospectively treated with infliximab (3 mg/kg). Changes in disease activity were monitored by the 28-joint DAS (DAS-28). Serum levels of different isotypes [immunoglobulins M, G and A (IgM, IgG and IgA)] of RF and anti-citrullinated peptide antibodies were measured by ELISA. RESULTS The mean DAS-28 decreased from 5.9 (1.1) at baseline to 4.0 (1.3) at Week 16 of infliximab treatment (P < 0.001). High baseline levels of different isotypes of RF (all P < 0.008), ACPA IgM (P = 0.008) and ACPA IgG (P = 0.07) were associated with an absolute decrease in DAS-28 after TNF blockade. This relationship persisted after adjusting for DAS-28 at baseline. However, the different isotypes of baseline RF and ACPA levels accounted for only a small proportion of variance in treatment response (RF: R² between 7 and 12% and ACPA: R² between 4 and 7%). The simultaneous presence of all three isotypes of RF or ACPA had no additive value. CONCLUSION Presence as well as the titres of RF and IgM ACPA at baseline are significantly correlated with better response to infliximab treatment. However, this correlation is not strong enough to allow a reliable prediction in individual patients. Trial Registration. ISRCTN Register, http://www.controlled-trials.com/isrctn/, ISRCTN36847425.

Lactoferrin and secretory IgA in the bronchoalveolar lavage fluid from patients with a stable asthma.

We have measured lactoferrin and secretory IgA (sIgA) in the unconcentrated bronchoalveolar lavage fluid (BALF) from nonsmoking healthy volunteers (n=10) and nonsmoking patients with stable asthma (n=14). The median concentrations and the ranges of lactoferrin were controls, 0.13 mg/L (0.01–0.43 mg/L); asthma, 0.41 mg/L (0.07–7.51 mg/L). For sIgA the results were controls, 0.48 mg/L (0.12–1.47 mg/L); asthma, 1.29 mg/L (0.65–14.6 mg/L). The concentrations in the epithelial lining fluid (ELF) were calculated on the basis of urea in BALF and serum. SIgA and lactoferrin levels in the BALF and ELF from the patients with asthma were higher than in controls (Mann-Whitney U-test, p<0.03).Our results indicate that in patients with stable asthma the airway epithelial cells are activated, resulting in an enhanced secretion of lactoferrin and enhanced secretory transport of sIgA into the airway lumen.

ELISA of complement C3a in bronchoalveolar lavage fluid.

Activated complement factors within the lung may induce several local biological effects. In order to investigate local complement activation we have developed non-competitive two-site ELISAs of C3a and total C3 in bronchoalveolar lavage fluid (BALF). For the assay of C3a, both C3 and C3(H2O) were removed from the samples by precipitation with polyethylene glycol. It was necessary to add carrier proteins to BALF to remove C3 and C3(H2O) fully. The ELISA of C3a has the lowest limit of detection reported thus far, namely 0.045 nM (= 0.405 ng/ml). In BALF from healthy persons (n = 9) the C3a concentration was 0.20 nM (0.12-0.31 nM) (median, range). C3a was higher in BALF from patients with asthma or with sarcoidosis; asthma (n = 10), 0.45 nM (0.20-5.79 nM); sarcoidosis (n = 19), 1.31 nM (0.095-5.65 nM) (Mann-Whitney U test, p less than 0.005). In BALF from patients with Pneumocystis carinii pneumonitis (n = 10) the C3a concentration was 0.18 nM (0.07-0.57 nM). C3a concentrations in BALF may reflect local complement activation in the lung and/or diffusion into the lumen. This was studied by normalizing C3a concentrations in BALF into values for epithelial lining fluid (ELF), and calculating serum-to-ELF quotients of C3a, and C3a/total C3 quotients.

Superinduction of Interleukin-6 mRNA in lung epithelial H292 cells depends on transiently increased C/EBP activity and durable increased mRNA stability

Restriction of eukaryotic protein synthesis affects the regulation of some transiently expressed gene transcripts resulting in their superinduction. We determined the transcriptional and post-transcriptional processes implicated in IL-6 mRNA superinduction in a human lung-derived epithelial cell line H292, and their kinetics in the absence and presence of an exogenous stimulus, tumor necrosis factor-alpha (TNF-alpha). Cycloheximide (CHI) at 10 microg/ml, which inhibited protein synthesis for 80%, caused a 80-fold induction of IL-6 mRNA level which was due predominantly to a stabilization of IL-6 mRNA (20-fold) early on. Employing transient transfection protocols we noted a small positive effect of CHI on transcription, mediated by the proximal and the distal C/EBP sites of the IL-6 promoter and paralleled by an increased C/EBP DNA-binding activity, similar to that found for exposure to TNF-alpha alone. TNF-alpha and CHI synergized on IL-6 mRNA expression (200-fold increase) which was due to an increased transcription, corresponding to a further increased C/EBP DNA-binding activity. However, the effect of CHI on IL-6 gene transcription was transient, in support of the need for ongoing protein synthesis for C/EBP activity. These findings indicate that IL-6 mRNA superinduction, at least in H292 cells, is regulated predominantly by modulating the repressive system that ensures a rapid degradation of IL-6 mRNA.

Characterization of CD4+ Memory T Cell Responses Directed against Common Respiratory Pathogens in Peripheral Blood and Lung

BACKGROUND We investigated CD4(+) memory T cell responses to influenza virus (FLU), respiratory syncytial virus (RSV), and nontypeable Haemophilus influenzae (NTHi). METHODS The precursor frequencies of antigen-specific CD4(+) cells were determined by in vitro expansion of peripheral blood mononuclear cells from healthy individuals (n=9) and patients with chronic obstructive pulmonary disease (COPD; n=16). The expression of CD27 and CCR7 and the production of interferon (IFN)- gamma and interleukin-2 was measured directly ex vivo. Furthermore, the phenotypic and functional properties of CD4(+) T cells residing in the lung were analyzed and compared with those of circulating CD4(+)memory cells from the same donors (n=8). RESULTS FLU-, RSV-, and NTHi-specific CD4(+) memory T cells circulated at low frequencies in the peripheral blood of healthy individuals and patients. RSV- and NTHi-specific CD4(+) T cells had a memory phenotype with moderate to high CD27 and CCR7 expression. In contrast to the low frequencies of circulating FLU-specific CD4(+) T cells, we found an enrichment of differentiated CD4(+) FLU-specific cells and high IFN- gamma expression in CD4(+) memory cells in lung tissue. CONCLUSION No gross defects were found in circulating CD4(+) memory cells specific for pathogens associated with COPD. However, the large differentiated CD4(+) memory T cell pool residing in the lung may contribute to a large extent to local antiviral immunological protection.