Subcutaneous immunotherapy suppresses Th2 inflammation and induces neutralizing antibodies, but sublingual immunotherapy suppresses airway hyperresponsiveness in grass pollen mouse models for allergic asthma.

BACKGROUND: Both subcutaneous and sublingual allergen immunotherapy (SCIT and SLIT) have been shown to effectively suppress allergic manifestations upon allergen exposure, providing long-term relief from symptoms in allergic disorders including allergic asthma. Clinical studies directly comparing SCIT and SLIT report a different kinetics and magnitude of immunological changes induced during treatment. Comparative studies into the mechanisms underlying immune suppression in SCIT and SLIT are lacking. OBJECTIVE: We aimed to establish an experimental model for grass pollen (GP) SCIT and SLIT that would allow a head-to-head comparison of the two treatments. METHODS: BALB/c mice were sensitized with GP extract, followed by SCIT and SLIT treatments with various GP dosages. Subsequently, we challenged mice with GP and measured airway responsiveness (AHR), GP-specific immunoglobulins, ear swelling tests (EST), eosinophilic inflammation in bronchoalveolar lavage fluid (BALF), and T cell cytokine release after restimulation of lung cells (IL-5, IL-10, and IL-13). RESULTS: We find that SLIT treatment was able to suppress allergen-induced AHR, while allergic inflammation was not effectively suppressed even at the highest GP dose in this model. In contrast, SCIT treatment induced higher levels of GP-specific IgG1, while SLIT was superior in inducing a GP-specific IgG2a response, which was associated with increased Th1 activity in lung tissue after SLIT, but not SCIT treatment. Interestingly, SCIT was able to suppress Th2-type cytokine production in lung cell suspensions, while SLIT failed to do so. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, GP-SCIT suppresses Th2 inflammation and induced neutralizing antibodies, while GP-SLIT suppresses the clinically relevant lung function parameters in an asthma mouse model, indicating that the two application routes depend on partially divergent mechanisms of tolerance induction. Interestingly, these data mirror observations in clinical studies, underscoring the translational value of these mouse models.

Shorter dosing intervals of sublingual immunotherapy lead to more efficacious treatment in a mouse model of allergic inflammation.

Current day practice of sublingual immunotherapy (SLIT) includes varying modalities of treatment that differ with regard to formulation, dosing and administration regimens. The aim of this study was to explore the importance of the dosing intervals in SLIT. The immunological effect of increased SLIT dosing frequency was tested in a mouse model of allergic inflammation. Mice sensitized to Phleum pratense (Phl p) were SLIT-treated with the same weekly cumulative dose administered with different administration frequencies. A SLIT sham-treated group was also included. All mice were challenged intra-nasally with Phl p extract following SLIT. Local and systemic cytokine production, eosinophil infiltration into airways and the development of Phl p-specific antibody responses were determined. Higher frequency of sublingual administration of allergen extract has a profound positive impact on the effect of SLIT, measured as induction of IgG and IgA antibodies. The once daily SLIT was the only treatment regimen being able to reduce all systemic Th2 cytokines and systemic IgE antibody responses when compared to sham-treated mice after the intra-nasal challenge period. The group receiving SLIT with the highest frequency of administration had the most pronounced effect of the treatment. In the same group, there was also a higher degree of protection against increase in IgE antibody levels after intra-nasal challenge with the allergen, our data demonstrate that a once daily regimen is more efficacious than regimens where SLIT, with the same weekly cumulative allergen dose, is administered with longer intervals but higher doses.

Sublingual immunotherapy reduces allergic symptoms in a mouse model of rhinitis.

BACKGROUND: Sublingual immunotherapy (SLIT) is a clinically effective treatment in both pollen and house dust mite-induced rhinitis and asthma. However, the mechanisms by which this is accomplished are not clear. OBJECTIVE: The objective of the current study was to establish a mouse model of rhinitis in order to study the effect and mechanisms of SLIT. METHODS: Mice were sensitized by intraperitoneal injections of alum-adsorbed Phleum pratense extract. Sensitized mice were SLIT-treated and subsequently challenged intranasally and analysed for clinical symptoms, antibody levels, eosinophilia and T cell response. RESULTS: Intranasal challenge of sensitized mice led to the development of rhinitis characterized by significantly increased sneezing and influx of eosinophils into the nose. Levels of specific IgE were fivefold increased in nasopharyngeal lavage (NAL) fluid and more than doubled in serum. Furthermore, a T-helper type 2 (Th2) like T cell response was observed in local draining lymph nodes. SLIT treatment of sensitized mice reduced sneezing, eosinophilia and IgE levels in the NAL by more than 50%. Moreover, serum levels of IgE and IgG1 as well as T cell response in the draining lymph nodes were also significantly reduced. Treatment for a shorter time or with a lower dose only led to minor reductions of the clinical and immunological parameters, indicating that the effect of SLIT is time and dose dependent. CONCLUSION: In the present study, we have established a mouse model displaying the hallmarks of allergic rhinitis using a clinically relevant allergen. Using this model, we have demonstrated that SLIT treatment is able to reduce allergic symptoms in a time- and dose-dependent manner.

CD4+ T regulatory cells from the colonic lamina propria of normal mice inhibit proliferation of enterobacteria-reactive, disease-inducing Th1-cells from scid mice with colitis.

Adoptive transfer of CD4+ T cells into scid mice leads to a chronic colitis in the recipients. The transferred CD4+ T cells accumulate in the intestinal lamina propria (LP), express an activated Th1 phenotype and proliferate vigorously when exposed ex vivo to enteric bacterial antigens. As LP CD4+ T cells from normal BALB/c mice do not respond to enteric bacterial antigens, we have investigated whether colonic LP-derived CD4+ T cells from normal mice suppress the antibacterial response of CD4+ T cells from scid mice with colitis. LP-derived CD4+ T cells cocultured with bone marrow-derived dendritic cells effectively suppress the antibacterial proliferative response of CD4+ T cells from scid mice with colitis. The majority of these LP T-reg cells display a nonactivated phenotype and suppression is independent of antigen exposure, is partly mediated by soluble factor(s) different from IL-10 and TGF-beta, and is not prevented by the addition of high doses of IL-2 to the assay culture. Functionally and phenotypically the T-reg cells of the present study differ from previously described subsets of T-reg cells. The presence of T cells with a regulatory potential in the normal colonic mucosa suggests a role for these cells in the maintenance of local immune homeostasis of the gut.

Impaired sensitivity to beta 2 integrin-blocking in ICAM-1-mediated neutrophil migration in ulcerative colitis.

BACKGROUND: Factors influencing the directed migration of neutrophils into colonic tissue in ulcerative colitis (UC) are poorly described. ICAM-1 has recently been shown to possess chemotactic properties, and the aim of this study was to evaluate the involvement of beta 2 integrins in this ICAM-1-mediated migration. METHODS: The chemotactic effect of ICAM-1 on neutrophils isolated from 13 UC patients and 17 healthy volunteers was studied in microchemotaxis chambers. Physiological concentrations of ICAM-1 (0.05-500 pM) were separated from neutrophils by nitrocellulose filters, and cell migration was evaluated using the leading front technique. beta 2 integrins on neutrophils were blocked with antibodies to CD11a, CD11b, CD11c and CD18, and migration towards ICAM-1 was examined. RESULTS: Migration towards ICAM-1 was equal for UC and control neutrophils, showing a bell-shaped ICAM-1 dosemigratory response curve with peak migration at 5 pM ICAM-1 (30.0 microns; interquartile range 22.9-35.7; P < 0.001). Blockade of the CD11 subunits on control cells inhibited the chemoattractant effect of ICAM-1 by 43.6%-58.0%, whereas the migration was decreased by only 20% in UC under similar blocking conditions (P < 0.01). Anti-CD18 mAbs had no effect. Inhibition of protein kinases with staurosporin only slightly decreased the ICAM-1-mediated migration, whereas incubation with staurosporin and CD11 antibodies showed additive effects on UC neutrophils and synergistic effects on control cells. No quantitative differences in beta 2 integrin expression were detected between control and UC neutrophils. CONCLUSIONS: The chemotactic property of ICAM-1 was shown to be CD11-dependent and UC neutrophils were found to be less dependent on CD11/ICAM-1-mediated migration than were control neutrophils.

Enteric bacterial antigens activate CD4(+) T cells from scid mice with inflammatory bowel disease.

Scid mice transplanted with CD4(+) T cells from congenic donor mice develop a chronic and lethal inflammatory bowel disease (IBD) 2-3 months post-transplantation. In the present study we have investigated the response of CD4(+) T cells from scid mice with colitis against fecal extracts. Our results show that in contrast to CD4(+) T cells from normal BALB/c mice, CD4(+) T cells from scid mice with colitis proliferate strongly in response to antigen-presenting cells (APC) pulsed with fecal extracts. The IBD-associated T cells did not respond to either extracts from food antigens or fecal extracts from germ-free mice, which indicates that they recognize bacterial antigens in the fecal extracts. CD4(+) T cells isolated from the colonic lamina propria of scid mice 3 weeks post transplantation also responded vigorously to fecal extracts, demonstrating that reactive CD4(+) T cells are present in the gut mucosa of transplanted scid mice prior to clinical manifestations of IBD. CD4(+) T cells activated by fecal extracts produced high amounts of IL-2 and IFN-gamma, intermediate amounts of IL-4 and low amounts of IL-10, consistent with a Th1 profile. The proliferative reactivity towards fecal extracts was restricted by MHC class II molecules and dependent on antigen processing, as the response could be blocked by anti-MHC class II antibodies or a short fixation of the APC. This study demonstrates that class II-restricted CD4(+) Th1 cells, which recognize enteric bacterial antigens, infiltrate the gut mucosa and spleen of transplanted scid mice prior to and during the course of colitis.

Immunoglobulin leakiness in scid mice with CD4(+) T-cell-induced chronic colitis.

Inflammatory bowel disease in scid mice is initiated by transplantation of CD4(+) T-cells from immunocompetent syngenic donor mice. As the disease progresses, immunoglobulin (Ig)-containing cells appear in the gut lamina propria, suggesting that locally accumulating Ig may play a role in disease development. In the present work we have investigated the relationship between disease progression and patterns or levels of Ig isotypes in the feces of scid mice suffering from an ongoing colitis. The data clearly showed that the severity or progression of the disease did not influence the levels of IgA, IgG1, IgG2a, IgG2b, and IgG3, whereas the level of fecal IgM increased during the course of colitis. The presence of the serum protein alpha-1-antitrypsin in fecal extracts from diseased mice suggests that some of the fecal Ig has leaked through the inflamed epithelial membrane into the gut lumen. Finally, Ig-containing cells were observed in mesenteric lymph nodes and in the spleen, suggesting that the fecal Ig is produced both systemically and locally in the gut wall. In conclusion, the present results demonstrate that the level of IgM increases as colitis progresses. Also, the five remaining major Ig isotypes are increased in the gut lumen of scid mice with colitis, but the individual Ig types vary randomly during the course of the disease. Thus, it is unlikely that immunoglobulins are involved in the immunopathogenesis of this model of colitis.

In vitro activated CD4+ T cells from interferon-gamma (IFN-gamma)-deficient mice induce intestinal inflammation in immunodeficient hosts.

To investigate the role of IFN-gamma in the immunopathogenesis of inflammatory bowel disease (IBD), severe combined immunodeficient (SCID) mice were transplanted with in vitro activated CD4+ T cells from either wild-type (WT) or IFN-gamma-deficient (IFN-gammaKO) BALB/c mice. In vitro, the two types of T cells displayed comparable proliferation rates and production of tumour necrosis factor-alpha (TNF-alpha), IL-2, IL-4 and IL-10 after concanavalin A (Con A) stimulation. When transplanted into SCID mice, WT CD4+ blasts induced a lethal IBD, whereas IFN-gammaKO blasts induced a less severe intestinal inflammation with moderate weight loss. Intracellular cytokine staining of lamina propria lymphocytes (LPL) revealed comparable fractions of CD4+ T cells positive for TNF-alpha, IL-2 and IL-10 in the two groups of transplanted SCID mice, whereas a two-to-three-fold increase in the fraction of IL-4-positive cells was found in IFN-gammaKO-transplanted SCID mice. Flow cytometric analyses showed strong up-regulation of MHC class II expression of colonic epithelial cells of WT-CD4+ T cell-transplanted compared with IFN-gammaKO-transplanted SCID mice. A significantly higher fraction of CD4+ LPL were found to enter the cell cycle, i.e. to incorporate bromo-deoxy-uridine, and to undergo apoptosis in vivo in WT-transplanted compared with IFN-gammaKO-transplanted SCID mice. These data point towards an important role for IFN-gamma in the development of IBD in SCID mice. The inflammation might be initiated and subsequently enhanced by the ability of IFN-gamma to induce de novo MHC class II expression in the colonic epithelium, a change which could lead to increased antigen processing and production of local proinflammatory cytokines, CD4+ T cell turnover and thereby to exaggeration of disease.

Reactions of N-N and N-O compounds with horseradish peroxidase and peroxidases from Mycobacterium tuberculosis.

Several N-N-and N-O-containing compounds were analysed for their ability to act as substrates for horseradish peroxidase and peroxidases in Mycobacterium tuberculosis extracts. Aminoguanidine, diaminoguanidine, isoniazid, hydroxylamine and hydrazine were found to be weak substrates for horseradish peroxidase in reaction I and to inhibit the reaction of horseradish peroxidase with hydrogen peroxide. The same compounds inhibited the reaction of Mycobacterium tuberculosis peroxidase-catalase with hydrogen peroxide, and hydroxylamine was found to be a weak substrate for this enzyme. In growth inhibition experiments, diaminoguanidine inhibited the growth of M. tuberculosis H37Rv at 50 microg/mL, but not the growth of two isoniazid-resistant strains. Isonicotinic acid hydroxamate inhibited the reaction of the peroxidases with hydrogen peroxide, but was not itself a substrate and had no growth-inhibitory effects. On the basis of these results we suggest that the effect of isoniazid on growth of M. tuberculosis results from increased oxidative stress due to inhibition of catalase-peroxidase as well as from generation of toxic radicals with the structure [structure in text].

Autoantibodies to molecular targets in neutrophils in patients with ulcerative colitis.

Autoantibodies against neutrophil granulocytes are frequently observed in patients with ulcerative colitis, but a precise description of the autoantigens involved is lacking. Therefore, sera from 75 patients with ulcerative colitis were studied for antibody specificities by means of indirect immunofluorescence microscopy, enzyme-linked immunosorbent assays, and immunoblotting of extracts of neutrophils, neutrophil granules and lymphocytes. Fifty-six percent of the sera reacted in indirect immunofluorescence with ethanol-fixed neutrophils. On formalin-fixed cells most sera shifted fluorescence pattern, indicating a cytoplasmic origin of antigen(s). Only a few sera reacted with specific, known antigens. Immunoblots showed reactions with a broad panel of antigens with preferences for proteins around 55-65 kDa, which were present in azurophilic granules and in cytosol, an 80-kDa protein found in the specific granules, and a 110-kDa unknown cytosol component. In conclusion, neutrophil-specific IgG autoantibodies from ulcerative colitis patients react with several different antigens, most of which are of nonnuclear origin.

Distinct differences in autoantigen specificity of anti-neutrophil cytoplasm antibodies in systemic vasculitides and other inflammatory diseases.

BACKGROUND: Anti-neutrophil cytoplasm antibodies in necrotizing vasculitides need to be distinguished from ANCAs in other inflammatory conditions to avoid clinical misinterpretation. OBJECTIVES: To help clinicians and laboratory scientists recognize and utilize vasculitis-related ANCAs as an aid in diagnostic workup and patient follow-up, and be aware that ANCAs with different characteristics are commonly found in other chronic inflammatory conditions that persistently engage neutrophils in the inflammatory process. METHODS: Indirect immunofluorescence and enzyme immunoassay methods were used to detect ANCAs with both known and unknown neutrophil autoantigenic targets. RESULTS: Primary necrotizing small vessel vasculitides such as Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, and renal-limited rapidly progressive necrotizing glomerulonephritis target either the serine protease proteinase 3 or myeloperoxidase in azurophilic granules. In ulcerative colitis and rheumatoid arthritis, we found multiple ANCA targets contained in azurophilic and specific granules, the cytosol and the nucleus, whereas PR3 and MPO were not, or only weakly, recognized. CONCLUSIONS: ANCAs typically found in active SVV are demonstrable both by indirect immunofluorescence and antigen-specific enzyme immunoassay, and strong reactivity to either PR3 or MPO is characteristic. Strong ANCA with MPO reactivity is also found in some patients with drug-induced syndromes (lupus, vasculitis). Intermediate to strong perinuclear ANCAs are found in a substantial proportion of patients with UC (40-60%) and RA (30-70%), but in these conditions the ANCAs have many antigen targets that are only weakly recognized.

Accumulation of immunoglobulin-containing cells in the gut mucosa and presence of faecal immunoglobulin in severe combined immunodeficient (scid) mice with T cell-induced inflammatory bowel disease (IBD).

Scid mice transplanted either with a gut wall graft or with low numbers of purified CD4+ T cells from immunocompetent syngeneic donor mice show clinical signs of IBD 3-4 months post-transplantation. The disease is mediated by mucosa-infiltrating CD4+ TCR alphabeta+ T cells. The pathology of 52 individual colon segments obtained from 20 gut wall- or CD4+ T cell-transplanted diseased scid mice was evaluated by histology and the numbers of infiltrating immunoglobulin-containing cells were determined. In particular, cells positive for IgM, IgA and non-inflammatory immunoglobulin isotypes such as IgG1 and IgG2b were found to accumulate in colon segments displaying the most severe histopathology, including inflammatory cellular infiltration, epithelial hyperplasia and ulcerative lesions. Compared with colon segments of normal C.B-17 mice, the lesional scid colon shows increased levels of cells positive for the IgG classes. Faecal extracts of the CD4+ T cell-transplanted scid mice revealed the presence of all six murine immunoglobulin isotypes. Disease progression was accompanied by an increased level of excreted IgM and IgG3 and decreased levels of IgA. It is concluded that locally secreted immunoglobulins may play an immunomodulating role in the pathological changes observed in the present model of T cell-induced inflammatory bowel disease.

Specificities of anti-neutrophil autoantibodies in patients with rheumatoid arthritis (RA).

The objective of this study was to characterize antigens recognized by neutrophil-specific autoantibodies from patients with RA. Sera from 62 RA patients were screened by indirect immunofluorescence (IIF). Positive sera were further tested by ELISAs for antibodies against various granule proteins and by immunoblotting of electrophoretically separated cell, granule or nuclear extracts. Forty-two sera (68%) reacted with ethanol-fixed neutrophils. In the ELISAs 32% of the 28 medium to strongly IIF-positive sera were negative, while 43% were weakly positive for more than one antigen. Immunoblots of whole neutrophils showed IgG reactions at 25-35 kD, in the 55-kD region, at 80 kD, and at 110 kD. Most sera reacted with more than one band. Except for the 55-kD antigen, none of the antigens appeared in lymphocytes. The most notable reactivity in subcellular fractions was with lactoferrin and with bands of 25-35 kD from nuclei. In conclusion, anti-neutrophil autoantibodies from RA patients recognize different antigens in the cytoplasm and in the nucleus. Lactoferrin is one of the common antigens recognized, but also unknown nuclear antigens of 25-35 kD mol. wt are involved.

Comparison of heparin-binding to lactoferrin from human milk and from human granulocytes by means of affinity capillary electrophoresis.

Human lactoferrin from two different sources: milk and secondary granules of neutrophil granulocytes, was compared with respect to heparin binding by means of affinity capillary electrophoresis. This method is based on analysis of migration pattern shifts induced by various amounts of ligand present in the electrophoresis buffer. Since lactoferrin is a basic molecule, analyses were performed at pH 8 in a capillary with a neutral hydrophilic coating. However, analyzable peaks were only obtained in the presence of heparin in the electrophoresis buffer. Proportionally to the amount of heparin present, these peaks exhibited shape changes and strong migration shifts. In the ensuing analysis it could be demonstrated that the two lactoferrins had comparable migration shifts and peak shape changes. This indicates that also in this respect the two forms of lactoferrin are identical. Affinity capillary electrophoresis is a convenient and fast method to investigate functional characteristics of low amounts of unmodified biomolecules.