S. Bonini

Nerve growth factor: an important molecule in allergic inflammation and tissue remodelling

Background: Several studies provide evidence that nerve growth factor (NGF) has an expanding role in neuroimmune interactions. Methods: We review our data on circulation levels of NGF in allergic diseases as well as on the relationships between this neurotrophin and primary and secondary effector cells of allergic inflammation. Results: In vernal keratoconjunctivitis, a close relationship exists between the increased NGF plasma values and the number of mast cells infiltrating the conjunctiva. NGF serum values are also increased in other allergic diseases and asthma, and are related to the severity of the inflammatory process and disease. Human CD4+ T cell clones (preferentially of activated Th2 type) produce and release NGF, and express high–affinity NGF receptors. NGF is preformed in and can act on human peripheral blood eosinophils to preferentially release inflammatory mediators. Immunoreactivity for high affinity NGF receptors is present both in basal epithelial cells and in the inflamed stroma of the allergic conjunctiva. Topical administration of NGF results in a complete healing of neurotophic corneal ulcers in man, thus suggesting a profound effect of NGF on human fibroblasts and extracellular matrix. Conclusion: Data presented suggest that NGF is an important molecule in allergic inflammation and tissue remodelling occurring in allergic diseases.

Animal models of allergic and inflammatory conjunctivitis

Allergic eye diseases are complex inflammatory conditions of the conjunctiva with an increasing prevalence and incidence. The diseases are often concomitant with other allergic diseases such as allergic rhinitis, atopic dermatitis and allergic asthma. Despite the disabling and prominent symptoms of ocular allergies, they are less well studied and further insights into the molecular basics are still required. To establish new therapeutic approaches and assess immunological mechanisms, animal models of ocular allergies have been developed in the past years. The major forms of allergic ocular diseases, seasonal and perennial allergic conjunctivitis, vernal and atopic keratoconjunctivitis and giant papillary conjunctivitis, each have different pathophysiological and immunological components. In contrast to these distinct entities, the current animal models are based on the sensitization against a small number of allergens such as ovalbumin, ragweed pollen or major cat allergens and consecutive challenge. Different animal species have been used so far. Starting with guinea‐pig models of allergic conjunctivitis to assess pharmacological aspects, new models including rats and mice have been developed which mimic major features of ocular allergy. The presently preferred species for the investigation of the immunological basis of the disease is represented by murine models of allergic conjunctivitis. In the future, combined ocular, nasal and aerosolic challenges with allergens may provide a model of allergy that encompasses simultaneously the target organs eye, nose and airways with conjunctivitis, rhinitis and asthma.

Conjunctival Provocation Test as a Model for the Study of Allergy and Inflammation in Humans

The clinical response after allergen challenge and immunologic mechanisms leading to tissue inflammation have been extensively studied in the skin, nose and lung of allergic subjects. The present paper reviews personal studies aimed at evaluating clinical, cellular and humoral events after administration of specific allergen to the eye. Specific conjunctival provocation tests performed in grass-sensitive patients caused persisting inflammatory changes in conjunctival scrapings and tear fluid with a significant accumulation of different inflammatory cells depending on the time of observation (neutrophils, 20 min; eosinophils, 6 h; neutrophils, eosinophils and lymphocytes, 12-24 h after provocation). Increasing the dose of allergen resulted in a dose-dependent increase in the number of inflammatory cells recruited. When high doses of allergen were used, the challenge not only induced late-phase histological changes, but also clinical symptoms 6-10 h after provocation. Several mediators of allergic inflammation, such as histamine, C3a des-Arg, leukotrienes B4 and C4, were also present and could be measured in tears after allergen challenge. Our studies represent the first evidence in humans that a late phase of allergic reaction occurs in the eye. They also suggest that the conjunctival provocation test may represent a model for the study of cells and mediators involved in the pathophysiology of allergic inflammation as well as of its pharmacologic modulation.

Conjunctival hyperresponsiveness to ocular histamaine challenge in patients with vernal conjunctivitis

We compared the conjunctival responsiveness to histamine diphosphate in patients with vernal conjunctivitis and in healthy control subjects. Fourteen asymptomatic patients with vernal conjunctivitis and 10 healthy volunteers were challenged in one eye with 10 microliters of increasing doses (0.01, 0.05, 0.5, and 1 mg/ml in phosphate-buffered saline) of histamine diphosphate. The contralateral eye was challenged with the diluent only. Photographs of each eye were taken for evaluation of conjunctival redness by two masked investigators. Both patients with vernal conjunctivitis and control subjects reacted to histamine with a dose-dependent conjunctival redness 2 to 5 minutes after ocular challenge. Conjunctival redness was more intense in patients with vernal conjunctivitis than in control subjects after ocular challenge with 0.01 and 0.05 mg/ml of histamine diphosphate solution (p less than 0.05). Moreover, the threshold concentration of histamine diphosphate, extrapolated from each individual dose-response curve, was significantly (p less than 0.02) lower in patients with vernal conjunctivitis than in control subjects. Our findings suggest that patients with vernal conjunctivitis demonstrate conjunctival hyperresponsiveness to a nonspecific challenging agent. Nonspecific conjunctival hyperreactivity, a novel concept in allergic eye disease, may be relevant for a better understanding of the pathogenesis and clinical variability of vernal conjunctivitis.

Effectiveness of nedocromil sodium 2% eyedrops on clinical symptoms and tear fluid cytology of patients with vernal conjunctivitis

A double-masked, randomised, placebo-controlled study was conducted to evaluate the effectiveness of nedocromil 2% eyedrops, a mast cell stabiliser, in 20 symptomatic patients with vernal conjunctivitis. A 1-week baseline period was followed by 6 weeks of treatment. Clinical examination and cytological evaluation of tear fluid were performed weekly, and the patients recorded their subjective assessment on a daily diary card. The nedocromil group showed significantly less hyperaemia in the course of treatment than did the placebo group, and significantly less itching at all visits compared with baseline itching. In the nedocromil-treated group, but not in the placebo group, the number of neutrophils, eosinophils and lymphocytes in tears decreased significantly during some treatment weeks when compared with baseline. The overall assessment of treatment efficacy by both clinician and patient was significantly in favour of nedocromil treatment over placebo.

Tear tryptase levels and allergic conjunctivitis

We measured tryptase, a neutral prolease stored in the secretory granules of mast cells, by solid‐phase radioimmunoassay in tears of 12 subjects with vernal keratoconjunctivitis (VKC) during remission phases, nine subjects with seasonal or perennial allergic conjunctivitis, and eight healthy controls. Mean values of tear tryptase levels were significantly (P<0.02) increased in VKC patients (14.5 ± 13 pg/l) when compared to those measured in patients with seasonal or perennial allergic con, junctivitis (0.6 ± 0.1 pgA) and in controls (3.3 ± 3.2 μg/l). In subjects with allergic conjunctivitis, the levels of tryptase, almost undetectable before allergen conjunctival challenge, showed a significant increase in the challenged eye 20 min ‐ but not 6 h ‐ after provocation in 5/9 cases. Our results indicate that VKC, a severe ocular disease characterized by an increased number and abnormal distribution of mast cells in the conjunctiva, also shows elevated levels of tryptase in tears even during remission phases. Evidence of mast‐cell activation, as revealed by a significant increase of tryptase levels in tears, is documented during the early‐phase reaction, but not during the late‐phase reaction, of allergic conjunctivitis patients challenged topically by specific allergen.

Control mechanisms of human basophil releasability

Extensive evidence suggests that mediator release from inflammatory cells plays a role in the pathogenesis of allergic disorders in humans.‘.2 Most studies have focused on the identification of new mediators de novo synthesized by human inflammatory cells; two such examples are AGEPC and leukotrienes.‘” In addition, a number of laboratories are striving to understand the immunologic and biochemical mechanisms underlying the release of chemical media-. tors.6-8 However, few studies have been devoted to clarifying the pathophysiologic mechanisms that control the release of mediators from human inflammatory cells (i.e., the concept of “releasability”). The concept and the study of releasability in humans originated in the laboratory of Lichtenstein and Conroy’ almost a decade ago. They foresaw that, in addition to quantitative immunologic parameters such as IgE and IgG antibody levels, the study of allergic disorders should include the evaluation of cellular releasability. Until then, releasability was defined on an almost philosophic plane because it was impossible to characterize and separate immunologic from releasability parameters. Schleimer et al.’ selected the basophil system, a reliable experimental model of human anaphylaxis, to initiate the study of releasability. For example, they showed that histamine release is not correlated to the absolute quantity of IgE antibody present on human basophils challenged with the appropriate antigen. 9 ” Similarly, the release of histamine in response to anti-IgE is not related to the number of IgE molecules on the basophil surface.” In addition, they demonstrated that significant differences in IgEand non-&E-mediated releasability exist in some allergic conditions.“-” These pioneering studies supported the notion that basophil releasability is a real entity that could be of some relevance in the pathogenesis of inflammatory disorders. Stimulated by the lack of information on the factors that affect basophil releasability, we initiated a series of investigations to identify and characterize factors that physiologically control basophil releasability. We also used the ba-

Studies on Human Basophil Releasability

Basophil releasability is an important parameter in several pathophysiological conditions. In normal donors, the maximum percent histamine release and cell sensitivity to rabbit anti-Fc epsilon (anti-IgE) is correlated with the age of cell donors. A positive correlation between serum IgE level and anti-IgE-induced histamine release was found in subjects below 20 years old. The response to formyl-Met-Leu-Phe (f-met peptide) was significantly reduced in subjects above 60 years old. In twins, IgE-mediated releasability and serum IgE levels appear to be controlled by two different genetic mechanisms. Basophils of patients with atopic dermatitis were more responsive than those of control subjects of matched ages to anti-IgE.

The eosinophil has a pivotal role in allergic inflammation of the eye

The pathophysiological role of eosinophils and their pivotal position in the network of inflammatory cells involved in allergic inflammation of the eye are outlined on the basis of several in vitro an

The pattern of the ocular late phase reaction induced by allergen challenge in hay fever conjunctivitis

The purpose of this study was to describe the pattern of the ocular late phase reaction in patients with ocular hay fever. The authors monitored the clinical and cytological conjunctival response after topical allergen challenge. A conjunctival provocation test was performed in six ryegrass-sensitive patients and six healthy volunteers with 320,000 Biological Units (Pharmacia, Uppsala) of ryegrass antigen, diluted in albumin, in one eye; albumin diluent alone was used in the second (control) eye. Clinical symptoms were evaluated before challenge after 20 min, then hourly for 12 hours and 24 hours after challenge. Cell counts in the tear fluid were performed at the same time interval The results obtained show a significant clinical reaction during the entire period, differing in peaks in individual cases. This reaction was associated with a persistent inflammation, with neutrophils in the early phase and eosinophils and lymphocytes in the late phase period The data thus show that ocular challenge with high allergen doses induces clinical and cellular evidence of an ocular late phase reaction and suggest that the conjunctival provocation test may be used for a better understanding of the pathogenesis of mild but persistent ocular inflammation in ocular hay fever.