Conjunctival mast cells and the allergic late phase reaction.

An active model of ocular anaphylaxis was developed in guinea pigs to evaluate the histopathology of the early (EPR) and late (LPR) phase reaction, focusing on the role of mast cells. Five groups (n = 6) of animals were actively immunized by first injecting into each of the axillary and inguinal lymph node areas, 0.25 ml of an emulsion containing 1 mg dinitrophenyl bovine gamma-globulin (DNP-BCG) with 0.5 ml complete Freund's adjuvant. After two weeks, an intramuscular injection of 0.5 ml of an emulsion containing 1 mg DNP-BGG with 0.5 ml of incomplete adjuvant was administered. One month after the first injection, animals were sacrificed after topical ocular challenge with 10 microliters of 1 mg/ml divalent hapten, di-DNP-lysine, in one eye and phosphate buffered saline (PBS) in the fellow eye as control. Clinical reactions were graded over time and histology evaluated at the endpoint (time 0, 0.5, 3, 9, and 24 h). Results showed that all animals clearly developed both an EPR and an LPR, as either a biphasic, multiphasic or prolonged clinical response. A small percentage of mast cells were degranulated at baseline, whereas, at 0.5 h, 95% of mast cells were degranulated in the eyes treated with specific hapten and 25% in the control eyes treated with PBS. At 3 h, 84% of the mast cells were degranulated. This value rose to 89% at 9 h, and remained unchanged at 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Conjunctival hyperresponsiveness to ocular histamine 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.

Clinical and cytologic aspects of ocular late-phase reaction in the guinea pig.

Conjunctival tissue of 12 guinea pigs (6 experimental animals, 6 controls) was sensitized with IgG1 (PCA titer 1:2,000) antisera to dinitrophenol carrier and challenged topically with hapten. Periorbital swelling, conjunctival edema and conjunctival redness were evaluated clinically at 0.5 h and hourly for 12 h, and again at 24 and 48 h. Tears were collected at each time point for cytology. Results showed an early-phase reaction (EPR) peaking at 0.5 h and a late-phase reaction (LPR) peaking at 5-7 h. Periorbital swelling was minimal 4 h after appearance of the EPR. Conjunctival edema and redness increased in both the early and late phases, but without two distinct peaks. However, individual animals in both groups did show biphasic reactions. Tear cytology showed an abnormal increase in neutrophils and eosinophils in the later time periods and was a significant way to detect ocular anaphylaxis. In conclusion, if the presence of two significant peaks of clinical inflammation after one antigen challenge is taken as the narrowest definition of a late phase in anaphylaxis, our results show an ocular LPR occurring in our animal model.

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 stabilizer, 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.

Vascular permeability during the early and late phases of ocular anaphylaxis.

The role of enhanced vascular permeability in a model of ocular anaphylaxis was investigated during both early- and late-phase reactions (EPR and LPR). Vascular permeability was assessed by measuring the extravascular retention of 125I-bovine serum albumin (125I-BSA) in ocular tissues. Ten groups of guinea pigs (n = 5-12 per group) were injected with dinitrophenylated (DNP) bovine gamma globulin emulsified in Freund's adjuvants and challenged after a 4-week interval by topical application of di-DNP-lysine to one eye and phosphate-buffered saline to the other eye. Thereafter, the eyes were examined and the animals were killed at different intervals after topical challenge. They were injected intravenously with 125I-BSA 0.5 hr before death. Retained radioactivity was measured separately in four tissues. The EPR (period between 0.5-1.5 hr after challenge) was characterized by enhanced retention of radioactivity in lids, conjunctiva, and orbital content. There was no significant retention of extravascular radioactivity in the globe and lacrimal gland. Thereafter (period between 2-3.5 hr after challenge), retained radioactivity was significantly diminished. The subsequent period, between 4.5-6.5 hr (LPR), was characterized by a smaller, although significant, increment of radioactivity retained in lids and conjunctiva but not in the other tissues examined. These findings indicate that enhanced microvascular permeability occurs during two phases in actively immunized guinea pigs challenged topically with di-DNP-lysine and that these phases correspond to the clinical signs that constitute the EPR and LPR of ocular anaphylaxis.

Tear volume in early and late phases of ocular anaphylaxis in guinea pigs.

Three models of ocular anaphylaxis containing 6 guinea pigs each were used to examine their effect on tear volume. In the first model, IgG1-rich anti-dinitrophenol (DNP) bovine gamma-globulin serum was injected subconjunctivally, and the animals were challenged 4 h later with topically applied di-DNP-lysine. In the second model, IgE-rich anti-egg albumin serum was injected subconjunctivally, and the animals were challenged 8 days later with topical instillation of egg albumin. In the third model, IgE-rich anti-egg albumin serum was injected intravenously, and animals were challenged 8 days later with topically applied egg albumin. Tears were collected before the procedure, 0.5 h after anaphylaxis was initiated, and then hourly for 8, 10, 12, 24, and 48 h after challenge. All animal eyes undergoing ocular anaphylaxis had significantly increased tearing. Tear volumes remained elevated in the first model beyond the observation period of 48 h.

Double hapten challenge in guinea pigs undergoing an ocular late-phase reaction.

Clinical and histological profiles of guinea pig conjunctiva undergoing late-phase reaction (LPR) were evaluated before and after additional antigenic challenge. Four groups of animals were passively sensitized with IgG1 antibodies, challenged with the specific hapten di-DNP-lysine, and rechallenged during LPR either with di-DNP-lysine, or with PBS, or with an aspecific antigen to characterize the reactivity of the inflamed conjunctival tissues. The course of the clinical anaphylactic responses was modified slightly only by challenge with a specific hapten during LPR. However, no significant changes in the inflammatory cell profile were observed in conjunctiva undergoing LPR after an additional challenge. This suggests the participation of mechanisms which may mediate the attenuation of the anaphylactic response in physiologic conditions of persistent antigen provocation.

Allergen dose response and late symptoms in a human model of ocular allergy.

Eleven ryegrass-sensitive patients were challenged weekly for 4 weeks with incremental doses of ryegrass allergen applied topically to one eye; a buffer was applied to the other eye. A clinical examination and tear-fluid cytology were performed before challenge and at 20 minutes, 1 hour, and 6 hours after challenge. A significant clinical reaction and neutrophil accumulation in the tear film occurred at 20 minutes. At 1 hour, a clinical response and tear cytologic reaction were present only at higher antigen concentrations. Six hours after antigen challenge, only the highest allergen concentration (320,000 BU/ml) produced a clinical late-phase reaction (LPR) (p less than 0.01) and tear cytologic change (presence of eosinophils and lymphocytes). Five nonryegrass-sensitive control subjects were unresponsive to a similar challenge. These results indicate that a conjunctival response to allergen challenge is dose dependent, that is, the higher the dose, the more likely an LPR will occur and that an LPR correlates with significant numbers of inflammatory cells in the tear film.

Influence of ocular surface antigen on the postnatal accumulation of immunoglobulin-containing cells in the rat lacrimal gland.

The objective of the present study was to determine whether antigenic presence on the ocular surface might directly influence the development or expression of the lacrimal secretory immune system. Experiments were designed to: (i) analyse the temporal accumulation of IgA-, IgG- and IgM-containing cells in lacrimal tissue during postnatal development (6-27 days of age); (ii) examine whether prevention of antigenic exposure to the ocular surface by unilateral tarsorrhaphy might inhibit lymphocyte immigration into the ipsilateral gland during development; and (iii) assess whether a non-invasive antigen, after placement on the ocular surface of infant or adult rats, undergoes retrograde transfer to the lacrimal gland. Our results demonstrated that: (i) the accumulation of IgA-, IgG- and IgM-containing cells in lacrimal tissue was most pronounced during the 6-day period after eyelid opening (15 days of age). The pattern of appearance of these Ig-containing cells, which were predominantly IgA-positive, was identical in both left and right lacrimal glands. (ii) Closure of the left lid by tarsorrhaphy from 10 to 18 days of age had no effect on the accumulation of IgA-, IgG- and IgM-containing cells in the left lacrimal tissue compared to cell numbers in the right gland. (iii) Following placement of radiolabelled albumin on the ocular surface, antigen was almost completely cleared within 1-2 hr. Analysis of lacrimal glands showed no significant accumulation of radioactivity at any time-point, either in the presence or absence of ocular inflammation. In contrast, up to 17.8% of radioactivity was found in the stomach 1-2 hr following topical antigen application. Overall, our results show that a rapid development of the lacrimal secretory immune system occurs between 15 and 21 days of age. This process does not appear to be dependent upon local antigenic stimulation. In addition, our findings indicate that a non-invasive antigen, when applied to the ocular surface, does not undergo retrograde transfer to the lacrimal gland. Instead, antigen appears to be cleared primarily through the gastrointestinal tract.

Early and late phases of ocular anaphylaxis in actively immunized guinea pigs.

A model of ocular anaphylaxis with distinct early- and late-phase components was studied in actively immunized guinea pigs. Twenty guinea pigs were injected with dinitrophenylated (DNP) bovine gamma globulin emulsified in Freund's complete adjuvant and challenged topically with di-DNP-lysine. Clinical signs were monitored over a 48 h period. An early-phase reaction (EPR) characterized by conjunctival edema, conjunctival erythema, lid swelling, and lid redness was observed. This reaction peaked at 0.5 h after challenge and subsided to a low point at 3-4 h. Subsequently, a second episode of lid swelling and lid redness was observed at 4-8 h. All animals in both groups exhibited an EPR. In addition, 75% of the animals underwent an EPR and an LPR. No animals exhibited an isolated LPR. Of the animals that underwent a dual response, 47% were biphasic, 6% were prolonged and 47% were multiphasic. The development of an active model of ocular anaphylaxis exhibiting both EPR and LPR components will enable studies of mechanisms which regulate the frequency and magnitude of these ocular allergic responses.

Age- and gender-related influence on the lacrimal gland and tears.

Previous research has demonstrated that distinct, gender-related differences exist in lacrimal glands in a variety of adult species. The objective of the current investigation was to examine whether this influence of gender extends throughout development and aging. Towards that end, morphological parameters of the lacrimal gland, as well as the volume, protein and IgG content of tears, were measured in male and female infant, preweanling, pubertal, adult, mid-life and senescent rats. Our results showed that dramatic, age-related variations occurred in the weight and morphological appearance of the lacrimal gland. Moreover, the magnitude of structural changes was dependent upon gender. Acinar area was significantly greater in lacrimal tissue of males, as compared to females, at all ages except pre-weanling; this sexual dimorphism was most evident in senescent rats. In contrast, acinar density in lacrimal glands of females was higher than, or equal to, that of males throughout development and aging. With regard to tears, pronounced increases were evident in tear volume, protein and IgG content from the pre-weanling stage to adulthood. After this time, these tear indices tended either to plateau or rise slightly until senescence. Gender had little or no impact of the volume of, or protein and IgG level in, tears. Of interest, the IgG/protein ratio in tears was 49- to 105-fold less than that found in lacrimal tissue, indicating that IgG moves down a gradient from the lacrimal gland to tears. Overall, our findings demonstrate that gender has a significant influence on lacrimal gland structure during development and aging. However, this impact is limited with respect to tear volume and protein content.

Penetrating the conjunctival barrier. The role of molecular weight.

Dinitrophenyl (DNP) derivatives of various molecular weights were tested for their ability to elicit ocular anaphylaxis after topical application to the eye of immunized animals. Adult male Sprague-Dawley rats were immunized by intraperitoneal injection of DNP-Ascaris suum extracts and alum and were then skin-tested with DNP-bovine serum albumin on day 13 post-immunization to assess their sensitivity to the DNP hapten. On day 14, animals were challenged topically with DNP derivatives in one eye; PBS was applied to the contralateral, control eye. Animals were evaluated clinically, and ocular tissues were processed for histologic evaluation. The compounds used for topical ocular challenge included the DNP derivative of egg albumin (MW 43,500 D), soybean trypsin inhibitor (MW 20,080 D), insulin (MW 5733 D), B-chain insulin (MW 3496 D), and lysine (MW 478 D). Only di-DNP-lysine elicited clinical signs of redness, edema, and tearing and histologic evidence of mast cell degranulation. None of the other compounds, tested in solutions of either equal numbers of milligram per milliliter or equimolar concentrations, elicited ocular anaphylaxis after topical application. A compound of low molecular weight, less than 3496, is needed to elicit ocular anaphylaxis when applied topically.

Clinical patterns of ocular anaphylaxis in guinea pigs passively sensitized with IgG1 antibody.

A model of topically induced ocular anaphylaxis in guinea pigs was developed. A guinea pig anti-dinitrophenyl (DNP) bovine gamma-globulin antiserum rich in IgG1 antibodies was injected subconjunctivally; various serum dilutions and latent periods were tested. The anaphylactic response was elicited with di-DNP-lysine applied topically. The response was characterized by rating 11 ocular signs. Although the 4-hour latent period is considered optimal for passive cutaneous anaphylaxis, a 14-hour latent period yielded the strongest ocular reaction and appeared to the most appropriate time for clinical assessment. Both an early-phase reaction (peaking in the 21st hour) and a late-phase reaction (peaking at 6-10 h) were observed. The two signs of the early-phase reaction that recurred in the late-phase reaction were periorbital swelling and lid redness. The anaphylactic reaction displayed different patterns in different animals: protracted, biphasic and multiphasic.

Determination of the interval during which one application of compound 48/80 to the rat conjunctiva influences the response to a second application.

We studied the effect of one application of compound 48/80 to the conjunctiva of the rat on the response to a subsequent challenge. Rats treated once showed conjunctival edema and marked degranulation of conjunctival mast cells. A second exposure to compound 48/80, applied after an interval of 24 h, produced slight clinical effects but had marked effects on conjunctival mast cells. Approximately 90% of the mast cells could no longer be observed by light microscopy. As the interval between initial challenge and rechallenge was increased, the clinical response returned, mast cells were again observable in normal numbers, and the extent of degranulation returned to approximately 50%. After an interval of 7 days, rats responded clinically as they had to the first application of compound 48/80. Although the number of mast cells in the conjunctiva was normal and extensive degranulation occurred, exocytosis was not observed. Thus some effects of the first application of compound 48/80 persisted for at least 1 week. Whether the effects observed depend upon the continued presence of compound 48/80 in the mast cell or granule membrane remains to be determined.

Neural-immune interrelationship: effect of optic, sympathetic, temporofacial, or sensory denervation on the secretory immune system of the lacrimal gland.

Previous research has demonstrated that the nervous system may exert a significant impact on lymphocyte distribution and function in certain mucosal tissues. Given this neural-immune interaction, the present study was designed to evaluate whether ocular nerves might influence the distribution, density, and output of IgA-containing cells in the lacrimal gland. Male Sprague-Dawley rats were subjected to one of the following surgical protocols: denervation of the temporofacial, optic, or trigeminal nerves or ablation of the superior cervical ganglion. Denervations were performed on one side and sham-operations on the contralateral side. Additional groups of rats were exposed to bilateral sham or complete optic nerve disruption. Two to four weeks after surgery, tears were collected from individual eyes for the measurement of IgA and protein levels and lacrimal glands were processed for the immunofluorescent identification of IgA-containing cells. Our results showed that a marked heterogeneity is present in the frequency distribution of IgA-containing cells in lacrimal tissue sections of sham-operated rats. Statistical analysis of this distribution showed that the topographical location of IgA-containing cells per section or through the gland was not random. This heterogeneity did not appear to be modified by interrupting various nerves to the eye. Moreover, severance of the temporofacial, optic, or trigeminal nerve supplies or extirpation of the superior cervical ganglion did not significantly alter either the density or total number of IgA-containing cells in the lacrimal gland, as compared to values in contralateral "sham-operated" tissues. In addition, these neural disruptions had no apparent influence on the IgA content or the IgA/protein ratio in tears.(ABSTRACT TRUNCATED AT 250 WORDS)

Histology of ocular late-phase reaction in guinea pigs passively sensitized with IgG1 antibodies.

The ocular late-phase reaction (LPR) is a mast cell-dependent, delayed inflammatory reaction developing 4-12 h after the early-phase reaction (EPR). We developed a passive IgG1 antibody-dependent guinea pig model that clinically reproduced the biphasic reaction of ocular EPR and LPR. An EPR was observed in all animals; a biphasic, multiphasic or prolonged inflammation was observed in the animals maintained for 9 and 24 h. The substantia propria of eyes undergoing EPR (0.5 h) showed intense edema, mast cell degranulation (88%), and 4-fold increase in eosinophils. At 9 h, the neutrophils and eosinophils had increased 11- and 25-fold, respectively. The number of basophils and lymphocytes was significantly increased compared to the controls (p less than 0.05). Of the mast cells, 45% were degranulated compared with 19% for controls. Cellular reactions had subsided by 24 h. Conjunctival epithelium also accumulated inflammatory cells as did the stroma of the lid skin. These histologic changes in ocular tissues undergoing anaphylaxis demonstrated that mast cell degranulation was most severe in the early phase, whereas neutrophil, eosinophil, and basophil accumulation was most marked in the late phase.

Effect of topical dexamethasone on the ocular allergic reaction in passively sensitized guinea pigs.

We examined the effect of extended topical pretreatment with dexamethasone (Dex) on the ocular allergic response in passively sensitized guinea pigs. The passive IgG1 antibody dependent guinea pig model used in these studies demonstrates both early- and late-phase ocular anaphylactic reactions. Fourteen hours prior to challenge with the hapten di-DNP-lysine, three groups of guinea pigs were sensitized subconjunctivally with antiserum rich in IgG1 anti-DNP antibodies in both eyes (group 1, n = 11) or in one eye (group 2, n = 9; group 3, n = 10). The contralateral eyes of animals in groups 2 and 3 received normal guinea pig serum. At 1-hour intervals, beginning 6 h prior to challenge, guinea pigs in group 1 received 0.1% Dex in 0.5% hydroxymethylcellulose (MC) topically in one eye and 0.5% MC in their other eye. Animals in group 2 were treated on the same schedule with 0.5% MC only in both eyes. Animals in group 3 were not pretreated. Conjunctival edema, conjunctival erythema, periorbital swelling, and lid erythema were scored independently in the upper and lower lids of both eyes of all animals for 10 h. Comparisons between the scores of Dex-pretreated eyes in group 1 and MC-pretreated eyes in group 2 or untreated eyes in group 3 revealed no significant differences in any clinical sign at 0.5 h (early-phase reaction). However, these comparisons demonstrated significant reductions in all four clinical signs in animals of groups 1 from 1 to 8 h and marked reduction or complete absence of signs from 6 to 10 h (late-phase reaction).(ABSTRACT TRUNCATED AT 250 WORDS)