Neutrophils contribute to inflammatory lymphangiogenesis by increasing VEGF-A bioavailability and secreting VEGF-D.

Lymphangiogenesis is an important physiological response to inflammatory insult, acting to limit inflammation. Macrophages, dendritic cells, and lymphocytes are known to drive lymphangiogenesis. In this study, we show that neutrophils recruited to sites of inflammation can also coordinate lymphangiogenesis. In the absence of B cells, intranodal lymphangiogenesis induced during prolonged inflammation as a consequence of immunization is dependent on the accumulation of neutrophils. When neutrophils are depleted in wild-type mice developing skin inflammation in response to immunization or contact hypersensitization, lymphangiogenesis is decreased and local inflammation is increased. We demonstrate that neutrophils contribute to lymphangiogenesis primarily by modulating vascular endothelial growth factor (VEGF)-A bioavailability and bioactivity and, to a lesser extent, secreting VEGF-D. We further show that neutrophils increased VEGF-A bioavailability and bioactivity via the secretion of matrix metalloproteinases 9 and heparanase. Together, these findings uncover a novel function for neutrophils as organizers of lymphangiogenesis during inflammation.

Attenuated Bordetella pertussis BPZE1 protects against allergic airway inflammation and contact dermatitis in mouse models.

BACKGROUND: We previously reported that prior nasal administration of highly attenuated Bordetella pertussis BPZE1 provides effective and sustained protection against lethal challenge with influenza A viruses. The protective effect was mediated by suppressing the production of major pro-inflammatory mediators. To further explore the anti-inflammatory properties of BPZE1, we investigated the effect of BPZE1 nasal pretreatment on two mouse models of allergic disease, allergic airway inflammation, and contact hypersensitivity (CHS). METHODS: Allergic reactions were induced in mice nasally pretreated with live attenuated BPZE1 bacteria using the ovalbumin (OVA)-induced allergic airway inflammation and dinitrochlorobenzene (DNCB)-induced CHS models. RESULTS: Prior BPZE1 nasal treatment suppressed OVA-induced lung inflammation and inflammatory cell recruitment and significantly reduced IgE levels and cytokine production. Similarly, BPZE1 nasal pretreatment markedly inhibited ear swelling, skin inflammation, and production of pro-inflammatory cytokines in the DNCB-induced CHS model. For both models, we showed that BPZE1 pretreatment does not affect the sensitization phase. Upon challenge, BPZE1 pretreatment selectively reduced the level of cytokines whose production is increased and did not affect the basal level of other cytokines. Together, our observations suggest that BPZE1 pretreatment specifically targets those cytokine-producing effector cells that are recruited and involved in the inflammatory reaction. CONCLUSION: Our study demonstrates the broad anti-inflammatory properties of the attenuated B. pertussis BPZE1 vaccine candidate and supports its development as a promising agent to prevent and/or treat allergic diseases.

A novel technique to explore the functions of bronchial mucosal T cells in chronic obstructive pulmonary disease: application to cytotoxicity and cytokine immunoreactivity.

Bronchial mucosal CD8(+) cells are implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, but there are few data on their functional properties. We have developed a novel technique to outgrow these cells from COPD patients in sufficient numbers to examine effector functions. Endobronchial biopsies from 15 COPD smokers and 12 ex-smokers, 11 control smokers and 10 non-smokers were cultured with anti-CD3/interleukin (IL)-2 ± IL-15. Outgrown CD3(+) T cells were characterized in terms of phenotype (expression of CD4, 8, 25, 28, 69 and 56), cytotoxicity and expression of COPD-related cytokines. Compared with IL-2 alone, additional IL-15 increased the yield and viability of biopsy-derived CD3(+) T cells (12-16-day culture without restimulation) without alteration of CD4(+) /CD8(+) ratios or expression of accessory/activation molecules. Biopsy-derived T cells, principally CD8(+)/CD56(+) cells, exhibited statistically significantly greater cytotoxic activity in current or ex-smokers with COPD compared with controls (P < 0·01). Elevated percentages of CD8(+) T cells expressed interferon (IFN)-γ, tumour necrosis factor (TNF)-α and IL-13 (P < 0·01) in current COPD smokers compared with all comparison groups. It is possible to perform functional studies on bronchial mucosal T cells in COPD. We demonstrate increased CD8(+)CD56(+) T cell cytotoxic activity and expression of remodelling cytokines in smokers who develop COPD.

CD8+ T cells in atopic disease.

In recent years there has been a tremendous expansion in our understanding about CD8(+) T cells. We now know that, as for CD4(+) T cells, they can be divided into subsets (Tc1 and Tc2) according to the cytokines they secrete. These subsets may differ in their capacity to kill and may even, in some cases, provide help for B cell antibody production or be involved in the induction of inflammatory responses. In addition, there is a host of cross-regulatory networks between different CD4(+) and CD8(+) subsets that control the magnitude and duration of immune responses. The observation that some antigens that are normally presented by MHC class II and seen by CD4(+) T cells can be presented by MHC class I and stimulate CD8(+) T cells increases the possibility for such interactions. During the next few years we can expect that our understanding of the biology of CD8(+) T cells and their role in immunity will increase.

Inhaled endotoxin in healthy human subjects: a dose-related study on systemic effects and peripheral CD4+ and CD8+ T cells.

BACKGROUND: Inhaled endotoxin or lipopolysaccharide (LPS) is implicated in the pathogenesis of pulmonary diseases. We investigated the inhalation effects of two different doses of LPS in healthy human subjects. METHODS: Eighteen healthy non-atopic human subjects inhaled either 15 microg (n=10) or 50 microg (n=8)Escherichia coli LPS in an open study. As control, each subject had isotonic saline inhalation 1 week before (baseline) and after LPS inhalation. Data collected included those of clinical parameter, induced sputum and peripheral blood CD4+ and CD8+ T cells. RESULTS: Acute flu-like symptoms and pyrexia were significantly greater in the 50 microg than 15 microg LPS group. Similarly, the increase in sputum and blood total cell and neutrophil counts at 6h following inhaled LPS were greater in the 50 microg group. Myeloperoxidase, human neutrophil elastase and interleukin-8 in sputum sol, but not blood, showed a trend towards greater increase following 50 microg LPS. All these changes were resolved at one week. In the 50 microg dose group alone, there was a reduction in the proportion of peripheral blood interferon (IFN)-gamma-producing CD4+ and CD8+ T cells at 6h followed by an increase at 1 week after inhaled LPS. CONCLUSIONS: The airway and systemic effects of inhaled LPS are dose-related and predominantly neutrophilic. The changes in the proportions of circulating CD4+ and CD8+ T cells suggests preferential recruitment of IFN-gamma-producing T cells into tissue from inhaled 50 microg LPS, followed by reappearance of these cells in blood 1 week later.

Activation-induced cell death of human T-cell subsets is mediated by Fas and granzyme B but is independent of TNF-alpha.

Human primary effector T cells were analyzed for their susceptibility to anti-CD3-induced activation-induced cell death (AICD). Th1 and Tc1 cells were more susceptible to AICD than their type 2 counterparts. Type 1 and type 2 subsets were also found to be differentially susceptible to CD95-mediated apoptosis, although cell-surface expression of CD95 and CD95L was at similar levels on all subsets. A role for CD95 in AICD was confirmed by the addition of anti-CD95L antibodies that partially abrogated AICD. Residual apoptosis could not be accounted for by TNF-alpha/TNFR interactions because although type 1 cells secreted more TNF-alpha than type 2 cells, the addition of TNFR:Fc fusion protein did not inhibit AICD. Instead, a reduction in AICD was observed in the presence of EGTA or concanamycin A. The inhibition of apoptosis by a granzyme B inhibitor z-AAD-CMK in Tc1 cells further indicated an involvement of the granule exocytosis mechanism in AICD.

Rapid washing of filter paper discs in a solid-phase radioimmunoassay with a constant flow washing device.

A machine has been developed for the rapid washing of the cellulose filter paper discs that are used in a number of radioimmunoassays. The machine is simple in design, easy to use, and is capable of washing 96 filter paper discs simultaneously. The efficiency of the machine is demonstrated by a RAST assay for measuring IgE antibodies to the venom. Time taken to wash the discs was reduced 3-fold without loss of sensitivity or reproducibility.

Increased speed and sensitivity, and reduced sample size of a micro-radioallergosorbent test (MRAST).

Performance of the radioallergosorbent test (RAST) in small volumes (MRAST) increases the efficiency with which both IgE antibody and 125I-anti-IgE bind. The rate at which antibody binds is increased up to 290-fold so that the assay can be completed in 60 min with comparable sensitivity to RAST. The increased speed of binding is achieved by the use of small incubation volumes which does not affect the rate at which bound antibody is dissociated. The resulting change in the equilibrium in favour of antibody binding yields a 10-fold increase in sensitivity which makes it possible to use as little as 5 microliter of sample compared with 50 microliter which is normal for RAST. MRAST can be used to measure IgE antibody in heel prick blood samples taken from babies, in rat sera and in other situations where the sample is in short supply.

Use of the enzyme-linked immunosorbent assay for measurement of allergen-specific antibodies.

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for allergen-specific IgG antibodies is described. Various solid-phase supports (microtiter plates), coating procedures, binding kinetics, and presentation of allergen in the assay were investigated. Using optimal conditions the indirect ELISA, in which the allergen is coated onto the well, was capable of detecting 2.4 ng/ml specific IgG antibodies to bee venom phospholipase A2(PLA2). The sandwich ELISA, in which the allergen was immobilized via specific antibody precoated onto the well, detected 0.24 ng/ml IgG antibodies to PLA2.

ELISA detection of human IgG subclass antibodies to Streptococcus mutans.

A sensitive enzyme-linked immunosorbent assay (ELISA) has been developed to measure IgG subclass antibodies against whole cells of Streptococcus mutans and to a purified streptococcal antigen (SA I/II). Bacterial cells were bound to the solid phase using methyl glyoxal and mouse monoclonal antisera against IgG and each IgG subclass were used to detect antibodies. Natural antibodies to S. mutans were predominantly of the IgG1 and IgG2 subclasses, though IgG3 and IgG4 antibodies were detectable in most subjects, and were the majority response in a few subjects. Antibodies to SA I/II were predominantly of the IgG1 subclass with virtually no activity detectable in the IgG3 and IgG4 subclasses. Inhibition studies suggested some restriction of IgG subclass responses to bacterial antigens since SA I/II and c polysaccharide could inhibit binding of all subclasses to whole cells of S. mutans equally, whereas glucosyltransferase, lipoteichoic acid and dextran showed greatest inhibition of the IgG3 and IgG4 subclasses.

Increased sensitivity and specificity of a sandwich ELISA for measurement of IgE antibodies.

We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for measurement of IgE antibodies to the bee venom allergens phospholipase A2 (PLA2) and hyaluronidase (HYAL). The assay is 10-20 times more sensitive than conventional indirect ELISA or radioallergosorbent test (RAST). Furthermore, by using affinity purified rabbit antibodies to these allergens, the specificity of the test was increased compared to RAST. The use of antibodies to link the antigen to the solid phase removes the dependence on the individual protein's ability to bind to the microtitre plate. The increased sensitivity of the sandwich assay seems to be due to better presentation and retention of antigen on the solid phase.

Development of a semi-quantitative enzyme-linked immunosorbent assay (ELISA) for detection of human IgG subclass antibodies.

We have developed a sensitive enzyme-linked immunosorbent assay (ELISA) which measures antibodies to bee venom phospholipase A2 (PLA2) and hyaluronidase (HYAL), horse IgG, bovine casein, and the bacterium Streptococcus mutans in each of the four human IgG subclasses. For this purpose, we have used mouse monoclonal antibodies (McAb) specific for each subclass and one which showed 'pan-IgG' reactivity. Binding to human IgG was similar for all the McAb and dilution of human IgG resulted in similar dilution curves for each subclass. Results were expressed as arbitrary U ml-1 by comparing the optical density obtained with each subclass-specific McAb to a reference curve for total IgG antibody constructed using the 'pan-IgG' McAb. Close agreement was found between the total amount of IgG antibody and the sum of the antibody in each of the four subclasses (PLA2 r = 0.90, horse IgG r = 0.98, bovine casein r = 0.84, S. mutans r = 0.85), confirming that these assays provide semi-quantitative measurements of the amount of subclass-specific antibody.

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) for the detection of picogram quantities of IgE.

Existing methods of measuring IgE in in vitro peripheral blood lymphocyte (PBL) cultures are not sufficiently sensitive to detect IgE when it is present in small amounts. This paper describes a modification of a two-site ELISA which increases the sensitivity of the assay 10-20-fold. By using the Fab' fragment of either rabbit or mouse monoclonal anti-IgE conjugated to alkaline phosphatase (AP) as the detector, the background of the assay was reduced sufficiently to permit signal amplification, using a commercially available amplified AP substrate. With this assay as little as 10 pg/ml of IgE could be detected. The interassay coefficient of variation was 15-18% between 1200 and 100 pg/ml IgE (n = 14) and there was a good correlation with a commercial IgE radioimmunoassay (RIA) (r = 0.98, n = 38).

The use of monoclonal and polyspecific antibodies in the IgE sandwich ELISA.

The use of antibody to link antigen to microtitre plates in the enzyme-linked immunosorbent assay (ELISA) has been extended to include mouse monoclonal antibodies and polyspecific rabbit antibodies. Small amounts of both reagents could be used. The capacity of the microtitre plate for the antibody was found to be critical and irradiated plates generally gave better results. Both rabbit anti-IgE conjugated to horseradish peroxidase, and monoclonal mouse anti-IgE with alkaline phosphatase-conjugated rabbit anti-mouse IgG could be used as detection reagents. Comparison with the radioallergosorbent (RAST) test showed a good agreement with the sandwich ELISA. The sandwich ELISA using polyspecific rabbit antibody was substantially more sensitive than conventional ELISA and also marginally more sensitive than RAST.

The purification of acid phosphatase from honey bee venom (Apis mellifica).

Acid phosphatase from bee venom was purified by a combination of saturated ammonium sulphate precipitation, gel filtration and ion exchange chromatography. The final product which is a glycoprotein contained less than 0.1% phospholipase A2 or hyaluronidase activity and existed in two molecular weight (96,000 and 45,000) forms. Acid phosphatase is a potent allergen, in bee venom allergic patients, which is capable of releasing histamine from sensitized human basophils and of inducing a wheal and flare reactions in sensitized human skin.

The effects of pollutants on the allergic immune response.

An increase in the prevalence of allergy and allergic diseases has taken place in the industrialised countries. Allergic diseases represent a major health problem, and appear linked to affluence and modern lifestyle. In the 20th century air pollution from industrial sources largely has been replaced by diesel exhaust and other traffic pollution. Further, the indoor environment in which we spend most of our time has changed dramatically. In order to understand the contribution of pollution and other environmental changes to the development of allergy, we need to understand the biologic processes that underlie allergic immune responses. In the present paper, immune regulatory pathways that control the allergic immune response are delineated. Castor bean dust causes widespread allergic sensitisation. The investigations that made clear the importance of CD8 T cells for the regulation of IgE production were triggered by studies of castor bean allergy. A special focus is in this review placed on the regulatory role of CD8 T cells in the development of the allergic immune response.

The role of the T follicular helper cells in allergic disease.

T follicular helper (Tfh) cells were discovered just over a decade ago as germinal centre T cells that help B cells make antibodies. Included in this role is affinity maturation and isotype switching. It is here that their functions become less clear. Tfh cells principally produce IL-21 which inhibits class switching to IgE. Recent studies have questioned whether the germinal centre is the main site of IgE class switching or IgE affinity maturation. In this review, I will examine the evidence that these cells are responsible for regulating IgE class switching and the relationship between Tfh cells and T helper 2 (Th2) effector cells.

Generation of a long-lived IgE response in high and low responder strains of rat by co-administration of ricin and antigen.

Certain strains of rats infested with the nematode parasite Nippostrongulus brasiliensis mount vigorous, persistent immunoglobulin E (IgE) responses. In the absence of parasites, adjuvants such as Bordatella pertussis or Al(OH)3 are needed to produce IgE responses to soluble antigens. These are short-lived, even in high IgE responder strains. In this study we have produced long-lived IgE responses in both low (Wistar) and high (Brown Norway) IgE responder strains of rats by repeated injections of ricin, a toxic lectin from castor beans, and phospholipase A2 (PLA2), a bee venom protein. Total IgE levels rose from 30 +/- 20 ng/ml to 39,000 +/- 7500 ng/ml in the Wistar rats compared with an increase from 120 +/- 100 ng/ml to 47,000 +/- 8000 ng/ml in the Brown Norway rats. An even greater (10(4)-fold) increase was seen in PLA2-specific IgE antibody levels. total and PLA2-specific IgE started to fall 6 weeks after treatment was stopped in the Wistar and after 12 weeks in the Brown Norway rats. The duration of the response was 204 and 248 days, respectively. The IgE-enhancing properties of ricin were compared in low, mid (Hooded Lister) and high IgE responder rats. Total IgE and PLA2-specific IgE but not IgG antibody (Ab) responses were enhanced in all animals given ricin and PLA2 but not in animals given ricin or PLA2 alone. The increase was greater in Wistar rats (48-fold) than in Brown Norway rats (eightfold) and by Day 24 the levels of both total and PLA2-specific IgE in three different strains were indistinguishable. PLA2-specific IgE antibody-secreting cells were detected in the spleen at a frequency of 1:5000. These results show: (i) that repeated immunization of rats with antigen and ricin produce a very large IgE response which was long-lived; (ii) that this response was indistinguishable in different IgE responder strains of rat; and (iii) that the IgE response declines earlier in low IgE responder strains of rats.