Mediators and Some Cytokines in Tears During the Late Conjunctival Response Induced by Primary Allergic Reaction in the Nasal Mucosa

The allergic conjunctivitis (AC) consists of five clinical types, a seasonal allergic conjunctivitis (SAC), perennial allergic conjunctivitis (PAC), vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC) and giant papillary conjunctivitis (GPC), having a common causal background, namely the involvement of allergic component, but different clinical features. 1-5 The five clinical types of AC can occur in 2 basic forms, a primary and a secondary form, with respect to the locality of the initial antigen-antibody/sensitized Th1 cells interaction with following steps, called initial allergic reaction. 6-12 In the primary AC forms, the initial allergic reaction with all subsequent steps, due to the direct exposure of conjunctivae by an external allergen, is localized in the conjunctival tissue. In these, classical, AC forms, the conjunctival tissue is the primary site of allergic reaction and together the primary target tissue affected directly by the allergic reaction and displaying the characteristic clinical symptoms. In the secondary AC forms, the initial allergic reaction taking place in the nasal mucosa, due to exposure to an external allergen, induces subsequently the secondary form of AC through various possible mechanisms and pathways. In this case, the conjunctival tissue is affected by factors released and generated by allergic reaction in the nasal mucosa and the conjunctival response displaying characteristic clinical symptoms may be considered as a consequence of the primary allergic reaction in the nose. 6-12 In both the basic forms of AC as well as all five clinical types, various hypersensitivity mechanisms, such as immediate type (IgE-mediated Type I), late (Type III) or delayed (cellmediated Type IV), may be involved.1, 2, 5, 9-19 The involvement of various hypersensitivity types in AC results then in development of various types of conjunctival response (CR) to allergen exposure (challenge), an immediate (ICR), a late (LCR), a dual late (DLCR, being a combination of an immediate and a late type), a delayed (DYCR) and a dual delayed (DDYCR, being a combination of an immediate and a delayed type). 13, 5-14, 20 The primary forms of AC can be demonstrated by direct conjunctival provocation tests with allergens (CPTs), whereas the secondarily induced AC forms can only be confirmed by nasal provocation tests with allergens (NPTs) in combination with registration of the conjunctival signs and subjective symptoms.


Introduction
The allergic conjunctivitis (AC) consists of five clinical types, a seasonal allergic conjunctivitis (SAC), perennial allergic conjunctivitis (PAC), vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC) and giant papillary conjunctivitis (GPC), having a common causal background, namely the involvement of allergic component, but different clinical features. [1][2][3][4][5] The five clinical types of AC can occur in 2 basic forms, a primary and a secondary form, with respect to the locality of the initial antigen-antibody/sensitized Th1 cells interaction with following steps, called initial allergic reaction. [6][7][8][9][10][11][12] In the primary AC forms, the initial allergic reaction with all subsequent steps, due to the direct exposure of conjunctivae by an external allergen, is localized in the conjunctival tissue. In these, classical, AC forms, the conjunctival tissue is the primary site of allergic reaction and together the primary target tissue affected directly by the allergic reaction and displaying the characteristic clinical symptoms. In the secondary AC forms, the initial allergic reaction taking place in the nasal mucosa, due to exposure to an external allergen, induces subsequently the secondary form of AC through various possible mechanisms and pathways. In this case, the conjunctival tissue is affected by factors released and generated by allergic reaction in the nasal mucosa and the conjunctival response displaying characteristic clinical symptoms may be considered as a consequence of the primary allergic reaction in the nose. [6][7][8][9][10][11][12] In both the basic forms of AC as well as all five clinical types, various hypersensitivity mechanisms, such as immediate type (IgE-mediated Type I), late (Type III) or delayed (cellmediated Type IV), may be involved. 1,2,5,[9][10][11][12][13][14][15][16][17][18][19] The involvement of various hypersensitivity types in AC results then in development of various types of conjunctival response (CR) to allergen exposure (challenge), an immediate (ICR), a late (LCR), a dual late (DLCR, being a combination of an immediate and a late type), a delayed (DYCR) and a dual delayed (DDYCR, being a combination of an immediate and a delayed type). 1-3, 5-14, 20 The primary forms of AC can be demonstrated by direct conjunctival provocation tests with allergens (CPTs), whereas the secondarily induced AC forms can only be confirmed by nasal provocation tests with allergens (NPTs) in combination with registration of the conjunctival signs and subjective symptoms. subjective symptoms (Tables 1, 2). The ocular signs and relevant subjective symptoms were evaluated by means of the Pelikan's scoring (grading) system (Table 3). 7,[9][10][11][12] The patients were investigated in a period without acute ocular and/or nasal complaints, without symptoms of an acute infection, outside the allergen-relevant period (season) and during. hospitalization. The long-acting H1-receptor antagonists, topical cromolyn and glucocorticosteroids were withdrawn 4 weeks, topical and oral short-acting H1 receptor antagonists, topical decongestants and other treatments were withdrawn 48 hours before each of the NPTs.

Patients
Control

Allergens
Dialyzed and lyophilized allergen extracts (Allergopharma, Reinbek, Germany) were diluted in phosphate-buffered saline (PBS) and used for skin tests in concentrations of 100-500 BU/mL and for NPTs in concentrations of 1000-5000 BU/mL (Table 2), as recommended by the manufacturer. If indicated, higher dilutions of the allergen extracts were used both for the skin tests and for the NPTs.

Skin tests
Scratch tests with allergenic extracts in concentrations of 500 BU/mL were performed and the results evaluated after 20 minutes. If the results were negative, then intracutaneous tests in concentrations of 100 BU/mL and 500 BU/mL were carried out and evaluated 20 minutes and 6, 12, 24, 36, 48, 56, 72 and 96 hours after the intradermal injection. A skin wheal (>7.0 mm in diameter) occurring after 20 minutes was qualified as a positive immediate skin response, the skin infiltration appearing between 6 and 12 hours as a late skin response, and the skin induration recorded later than 48 hours as a delayed skin response. 8-13, 42-46

Nasal provocation tests (NPTs)
Nasal challenges with allergens were performed using rhinomanometry, already described in our previous studies. 6-13, 23, 43-47 The nasal obstruction due to the edema of the nasal mucosa was evaluated by means of nasopharynx-nostril pressure gradient (NPG) parameters, which are the pressure differences (∆P) between the nasopharyngeal cavity and the outside air, expressed in cm H 2 O. NPTs were performed using the following schedule: (1)  The NPG changes recorded within 60-120 minutes after the allergen challenge were considered to be an immediate NR (INR), those recorded within 4-12 hours to be a late NR (LNR), and the changes measured later than 24 hours to be a delayed NR (DYNR) 9-13, . 43-45

Control tests with phosphate-buffered saline (PBS)
The control nasal challenge with PBS was performed in each patient studied by the same schedule as that used for the NPTs with allergen, however 3 days later.

Conjunctival response
The objective conjunctival signs and relevant subjective symptoms were registered before and during all NPTs with allergens and PBS at the same time-points as the nasal NPG values. The features of the conjunctiva were assessed by ophthalmoscopy including a slit lamp. The conjunctival signs, hyperaemia (injection), chemosis, hyperlacrimation, and palpebral edema, and the subjective symptoms, such as itching (burning), blurred vision and photophobia, were registered and evaluated by means of the scale suggested by Abelson, however, modified by us (Pelikan's scale). [10][11][12] The evaluation criteria of the individual signs and symptoms were as follows: 0=absent, 1=mild (present to a slight degree), 2=moderate, 3= pronounced (moderately severe), 4=severe ( Table 3 ). The differences in total sign score of 4 points or more ( 3 ± 1, mean ± SE), recorded at least at three consecutive time-intervals, were found to be statistically significant (p<0.05).

Collection and processing of tears
The tear specimens were collected from each of eyes separately by means of a micropipette from the inferior conjunctival fornix and/or lacus lacrimalis, before, 30 and 60 minutes, every second hour up to 12 hours and 24 hours after the allergen challenge. If necessary, a gentle pressure on the lacrimal sac from outside was applied. The tear samples (1-4 mL ) were stored at -8ºC and processed within 1 hours. The concentrations of appropriate factors in tears were measured by using commercially available kits, following the manufacturer's recommendations. The measurements of the factors were performed separately in tear samples from each of the eyes on each occasion, and the results were then calculated as the mean of both the eyes. All measurements were performed in duplicate by a double-blind schedule. The intraassay as well as the inter-assay coefficients of variations for all the assay kits employed were less than 10 %. a. Histamine Histamine concentrations, so-called "blanks", were measured by the Siraganian's fluorometric method 48

Control group
Fourteen adults suffering from allergic rhinitis, confirmed by positive history, skin tests and positive NPTs with inhalant allergens, but without history of any ocular disease and with normal ophthalmologic findings, volunteered to participate as control subjects. In these patients 14 positive late nasal responses (LNR)with inhalant allergens were repeated and supplemented with registration of the conjunctival features and subjective symptoms and estimation of the above mentioned mediators in tears. Statistical evaluation of the CR was performed separately for each of the eyes and then the mean from both the eyes was calculated. A P value < 0.05 was considered to be statistically significant.

Nasal responses (NRs)
In the 31 patients 54 nasal provocation tests (NPTs) with various inhalant allergens (Tables 1, 2) and 31 PBS control challenges were performed. The 31 patients developed 31 late nasal responses (LNRs; p<0.01) and 23 negative nasal responses (NNRs; p>0.1)( Table 2). The LNR began between 4-6 hours, reached its maximum between 6-8 hours and resolved within 12 hours after the nasal challenge with allergen. The 31 PBS control tests were all negative (p>0.1). No significant differences were found in the appearance of the LNRs with respect to the individual allergens (p>0.1). The LNRs were associated with significant changes (p<0.05) in the counts (mostly temporary increase) of the neutrophils, eosinophils, epithelial and goblet cells, and to a lesser degree of the lymphocytes, in the nasal secretions. The counts of basophils, mast cells, monocytes and plasma cells were relatively low and without significant changes. The repeated NPTs resulted in the development of similar and statistically significant LNRs as comparing the post-challenge with the pre-challenge (baseline) values (p<0.001) and with the PBS control values (p<0.001) (Fig. 1C ). No statistical significant differences were found between the initial and the repeated LNRs (p>0.2).

Conjunctival responses (CRs)
The 31 positive LNRs, recorded in 31 patients, were associated with significantly positive secondary conjunctival responses of the late type (SLCR; p<0.01)( Table 2). The positive SLCR began between 5-6 hours, reached its maximum between 8-10 hours and resolved usually within 12, sometimes within 24 hours after the allergen challenge. The SLCR was represented by significant changes of the objective conjunctival signs (p<0.01) as well as subjective symptoms (p<0.05). No significant corneal signs were recorded in any SLCR.
No conjunctival changes were recorded during the 23 negative nasal responses (p>0.05) or during the 31 PBS control challenges (p>0.1). The 31 repeated NPTs , have induced similar and statistically significant SLCRs , both as comparing the post-challenge with the prechallenge (baseline) values (p<0.01) and as comparing with the PBS control challenge (p<0.01). (Fig. 1B). No statictically significant difference were found between the initial and the repeated SLCRs (p>0.2). No significant differences in the conjunctival changes recorded both during the initial and during the repeated SLCRs were observed between the right and left eye (p>0.1).

Changes of mediators and other factor in the tears during the SLCRs
The SLCRS were associated with significant changes in the concentrations (p<0.05) of histamine, EPC, LTC 4 , LTB 4 , MPO, IL-4 and IL-5 in the tears (Table 4; Fig. 1A). The prechallenge concentrations of most of these factors were either very low or under the detection limit, whereas their post-challenge concentrations usually increased to various degrees at various time-points, followed by their decrease and disappearance from the tears within 24 hours after the allergen challenge (Table 4 ; Fig.1A). The concentrations of tryptase in tears was very low, sometimes under the detection limit and without significant changes (p>0.05). The INF-γ and IL-2 were recorded in the tears during the SLCRs irregularly and without any significant changes (p>0.05 and p>0.05, respectively). The LTE 4 was not detected in the tears during the positive SLCRs. No significant concentration changes of the investigated factors were recorded in tears during the 31 PBS control challenges and 23 negative CRs. Moreover, the concentrations of most of these factors were under the detection limits (Table  4). No significant differences in the concentrations of particular factors and their changes in tears have been found between the right and left eye, both during the SLCRs and during the PBS controls (p>0.1 and p>0.2, respectively).
www.intechopen.com      Table 4. Concentration of particular factors in tears during the secondary late conjunctival response (SLCR)

Control patients
The 14 control patients developed an isolated late nasal response (ILNR) during the initial as well as the repeated NPTs (p<0.001 and p<0.001 respectively). No significant conjunctival signs or subjective symptoms were recorded during the 14 repeated LNRs (p>0.2). No significant changes in concentrations of the investigated factors were recorded in tears during the repeated LNRs in these control patients.

Discussion
The relationship between the conjunctivae and the nose includes both the anatomical and the functional aspect. 1,[10][11][12] The conjunctiva is connected with the nasal cavity not only by means of the naso-lacrimal duct, being a part of lacrimal ways, through which opening the tear drainage into the nasal cavity is facilitated, but also by means of the blood vessel network, lymphatic tissue system and neurogenic network. All of them express a number of mutual links and share various common properties. 1, 10-12 Allergic reactions taking place primarily in the nasal mucosa due to the intranasal exposure to inhalant allergen may affect the conjunctiva and subsequently also other ocular tissues, such as the cornea, in various ways and upon involvement of various mechanisms. 9-12, 33, 49-58 These mechanisms may include: (1) Various cell types participating in the allergic reaction occurring in the nasal mucosa, such as mucosal mast cells, eosinophils, basophils, neutrophils, Blymphocytes/plasma cells, particular subsets of T-lymphocytes (Th`1, Th2, Th 17, Tregulatory cells, natural killer cells), dendritic cells, monocytes thrombocytes, macrophages, epithelial and endothelial cells, and mucosal goblet cells, can migrate into the bloodstream and/or lymphatic system, and under extreme conditions also into lacrimal ways, and thereby attain the conjunctiva; (2) The various cell types, activated and/or inhibited during the allergic reaction in the nasal mucosa, generate and release a number of factors (classical mediators, eicosanoids, cytokines, chemokines, adhesion molecules, chemotactic and other factors), which could then reach conjunctiva either directly by the retrograde penetration through the naso-lacrimal duct and lacrimal ways or indirectly through the related blood and/or lymphatic vessel system; (3) The allergic parameters before and repeatedly after the allergen challenge, thus for a sufficiently long period of time, allowing measurement of the particular response type in its whole and dynamic course.