Veterinary immunology

Veterinary Immunology and Immunopathology 44(1995) 115-128

and immunopathology

__~

Platelet activating factor mimics antigen-induced cutaneous inflammatory responses in sweet itch

horses

A.P. Foster, P. Lees, F.M. Cunningham Department of Veterinary Basic Sciences, The Royal F’eterinary College, Hawkshead Law.

North Mymms ALY 7TA, UK

Accepted 1 February 1994

Abstract

Hypersensitivity responses to biting flies such as Culicoides are believed to be the cause of sweet itch, a seasonal intensely pruritic skin condition of horses. Little is known about the mediators released by antigen in the skin of affected horses. In the present study the cutaneous vascular and cellular responses to intradermally injected platelet activating fac- tor (PAF) have been characterised in sweet itch cases during the active phase of the dis- ease and compared with those of Culicoides antigen extract. Histamine was used as a pos- itive control in vascular permeability studies. Responses were also examined in 4 of the 5 sweet itch cases during the inactive phase of the disease. Norma1 ponies were used as controls.

PAF-induced increases in vascular permeability that were dose-related (0.00 1- I pg per site) and of a similar magnitude in sweet itch and normal animals. Antigen (O.S-5Opg per site) also caused dose-related wheal formation in sweet itch cases during the active, but not the inactive, phase of the disease. This effect was biphasic, with maximal responses occurring at 1 and 8 h. An increase in vascular permeability occurred in normal ponies only after administration of the highest dose of antigen tested. Interestingly. histamine (O.OZpg per site) induced wheals were significantly smaller in the affected. compared with the normal, group, both during the active and inactive phases.

PAF and antigen caused neutrophil accumulation in the skin of sweet itch and normal animals during both the active and inactive phases of the disease. Eosinophil recruitment was also observed but only in the affected group and, in the case of PAF. during the active, but not the inactive, phase. Antigen additionally caused the accumulation of mononuclear cells in the skin of sweet itch cases during the active phase, PAF induced a small increase in mononuclear cell numbers in these animals but the increase was not statistically significant.

*Corresponding author. Fax 0707 652090.

0165-2427/95/$09.50 @ 1995 Elsevier Science B.V. ,411 rights reserved .%SDIOI65-2427(94)05299-8

116 A.P. Foster et ai. / Veierinary Immunology and Imi~u~o~a~~ology 44 (I 995) I IS- I2S

These findings demonstrate that PAF mimics the effects of Culicoides antigen during the active phase of the disease. Hence, PAF, like histamine, may play a role in the patho- genesis of antigen-induced responses in the skin of sweet itch horses.

Abbreviations

AUCs, area under the curves; i.d., intradermal; Ig, immunoglobulin; ESA, equine serum albumin; PAF, platelet activating factor; PBS, phosphate buffered saline; sem, standard error of mean.

1. Introduction

Sweet itch is an intensely pruritic seasonal skin condition of horses typically occurring between April and October in the United Kingdom, and affecting up to 3% of the population (McCaig, 1973; Mellor and McCaig, 1974). Evidence suggests that this disease is an hypersensitivity to biting flies, in particular those of the genus Culicoides and antigen-specific immunoglobulin (Ig) E-like anti- body has been detected in sweet itch cases (Mellor and McCaig, 1974; Matthews et al., 1983). The primary lesions are papules; later, crusting, serous effusion, excoriation, broken hairs and alopecia are evident (Fadok, 1987; Scott, 1988). Histologically there are variable degrees of superficial and deep perivascular der- matitis. The leucocytic infiltrate is mixed, containing predominantly eosinophils and lymphocytes (Fadok, 1987).

Studies have been carried out to characterise the response of hypersensitive horses to antigen using aqueous Culicoides extracts (Reik, 1954; Baker and Quinn, 1978; Quinn et al., 1983; Larsen et al., 1988; Fadok and Greiner, 1990). How- ever, little is known about the identity or effects of inflammatory mediators re- leased in equine skin following antigen challenge. Histamine has been implicated in the pathogenesis of sweet itch (Reik, 195 5 ), although anti-histamines are rarely beneficial in the treatment of this disease (Scott, 1988). Studies in man have suggested that platelet activating factor (PAF), like histamine, induces pruritus and may play an important role in the later stages of allergic cutaneous responses (Henocq and Vargaftig, 1988; Roberts et al., 1988; Shalit et al., 1989). Recent work in normal horses has shown that intradermal injection of PAF increased cutaneous vascular permeability and caused dermal neutrophil accumulation (Foster et al., 1992a). Furthermore, in vitro studies have demonstrated that PAF is a chemoattractant for equine eosinophils in addition to neutrophils (Foster et al., 1992b).

The aim of the present study was to investigate whether PAF mimics the effects of antigen by increasing cutaneous vascular permeability and causing inflamma- tory cell accumulation in the dermis of sweet itch cases. Since the cutaneous in- flammatory responses of affected animals caused by PAF and antigen might be altered by the level of natural antigen challenge, the effects of PAF and antigen were compared during the active and inactive phases of the disease.

A.P. Foster et al. / Veterinary Immunology and Immunopathology 44 (1995) 115-128 117

2. Methods

2.1. Materials

Stock solutions of C 16 : 0 PAF and lyso-PAF ( 1 mg ml-’ in ethanol; Bachem, Saffron Walden, UK) were prepared and stored at - 20°C until required. After evaporation of ethanol under nitrogen, PAF and lyso-PAF were dissolved and diluted in phosphate buffered saline containing 0.25% equine serum albumin (PBS/ESA) for vascular permeability studies and in PBS alone for cell accumu- lation studies. Histamine diphosphate (2 mg free-base ml-’ in sterile PBS; Sigma Chemical Co., Poole, UK), the positive control used in vascular permeability studies, was stored at 4 “C and dilutions prepared in PBS/ESA to facilitate direct comparison with PAF. A whole body extract of Culicoides ( 1 mg ml- ’ in sterile PBS) (cAg ), containing equal amounts of Culicoides obsoletus, Culicoides dwelfi, Culicoides punctatus and Culicoides pulicaris was kindly supplied by Professor K. Baker (University Veterinary School, Republic of Ireland ). Aliquots were stored at -20°C and further dilutions made in PBS. Dilutions of PAF, lyso-PAF, his- tamine and antigen were freshly prepared under sterile conditions when required and administered by intradermal (i.d. ) injection in 50 ~1 volumes.

2.2. Animals

Two castrated male and two female Exmoor ponies, as well as one castrated male hunter, with clinical histories of seasonal recurrent pruritus, consistent with the diagnosis of sweet itch, were used in this study. Under field conditions clinical signs were evident between May and November (active phase of the disease). From December to April, lesions resolved and the cases were not pruritic (inac- tive phase of the disease). Six control ponies were taken from a group of healthy animals comprising six castrated male New Forest and two female Welsh moun- tain ponies. All animals were treated with anthelmintics at regular intervals and routinely vaccinated against tetanus.

Twenty-four hours prior to each experiment normal areas of skin on the dor- solateral aspects of the necks of sweet itch and of normal animals were clipped and shaved.

2.3. Effects ofmediators and CAg on cutaneous vascular permeability and inflammatory cell accumulation during the active phase of the disease

2.3.1. Vascular permeability PAF (0.001-l pg per site) and the vehicle (PBS/ESA) were administered to

sweet itch and normal animals. Histamine (0.02 and 0.2 pg free-base per site) was used as a positive control. Previous studies have shown that lyso-PAF, the precursor and metabolite of PAF reported to be biologically inactive in other spe- cies, has no effect on vascular permeability in the skin of normal ponies (Foster et al., 1992a). Hence, lyso-PAF (1 pg per site) was injected only in sweet itch

118 A.P. Foster et al. / Veterinary immunology and ~rnt~~nop~t~o~og~l44 (I 995) I IS- 128

cases as a negative control. In a second study, CAg (0.0005-50 ,ug per site) and the vehicle (PBS) were administered to sweet itch and normal animals.

Measurements of skinfold thickness were made prior to and between 4 and 64 min after administration of the mediators, using spring guage calipers and two perpendicular wheal diameters measured using vernier calipers. Measurements were taken before and between 0.25 and 24 h after injection of antigen. Changes in vascular permeability have been calculated as wheal volumes, the shape of which closely approximated to half an ellipse, using the formula 2/3z(r,.r,.r,) where r, and r, represent the 2 wheal radii and r3 is the increase in skinfold thickness.

2.3.2. Injlammatory cell accumulation Six millimetre punch biopsies were removed under local anaesthesia (2% lig-

nocaine without adrenaline; C-Vet, Bury St. Edmunds, UK) 2, 8 and 24 h after administration of PAF ( 1 pg per site), CAg (5 pg per site) and vehicle (PBS) to sweet itch cases. In normal ponies biopsies were taken at the same time points following injection of CAg and PBS or 2 and 24 h after administration of PAF. Biopsies were also removed from the untreated skin of sweet itch and normal animals.

After fixing biopsies in modified formaldehyde saline solution, 4 pm thick par- affin sections were cut and stained with Giemsa. The numbers of neutrophils, eosinophils and mononuclear cells in 4 mm* of deep dermis were counted.

2.4. Effects of mediators and CAg on cutaneous vascular permeability and inflammatory cell accumulation during the inactive phase of the disease

2.4.1. Vascular permeability The effects of histamine (0.02-0.2 fig per site) and CAg (0.005-5 pg per site)

on cutaneous vascular permeability were examined in four of the sweet itch cases as described above. The effects of PAF were not investigated as no differences were obtained between the permeability responses of affected and normal ani- mals during the active phase (Fig. 1 (a) ).

2.4.2. Inflammatory cell accumulation The effects of PAF ( 1 pg per site), CAg (5 pg per site), and the vehicle (PBS)

on cell accumulation were examined at 2 and 24 h.

2.5. Peripheral blood leucocyte counts

Venous blood samples were obtained from normal and sweet itch animals dur- ing both the active and inactive phases of the disease. Total peripheral blood leu- cocyte counts were carried out using a Coulter counter and differential counts performed on at least 200 cells.

‘4. P. Foster et al. / Veterinary immunology and I~t~rrl~~noputhologv 44 (1995) 1 i_S- 128 119

(a)

500 -

n,

.OOl -01 1 1 PAF @g/site)

(b)

800

1 l-

0 02 0.2

Histamine (pgisite)

Fig. 1. Effect of (a) PAF and (b) histamine on cutaneous vascular permeability 32 min after intrad- ermal injection in (m) sweet itch (n = 5) and (0 ) normal (n = 6) animals during the active phase of the disease. Each point or bar is the mean of values obtained after subtraction of the vehicle response. Vertical lines represent sem*= P-c 0.05 compared with normal ponies.

2.6. Statistical analyses

Results are expressed as the mean 2 standard error of the mean (sem ) after subtraction of the response to vehicle alone. Where appropriate, areas under the curves ( AUCs) were calculated by the trapezoidal method. Dose-related effects were demonstrated by the transformation of data to natural logarithms and anal- ysis of regression. Statistical analyses were carried out using paired or unpaired Student’s t-tests for single comparisons, or by a Dunnett’s test for multiple com- parisons. A P value of less than 0.05 was considered to be statistically significant.

3. Results

3. I. Effects of mediators and CAg on cutaneous vascular permeability during the active phase of the disease

PAF (0.00 1- 1 pg per site) produced a dose-related increase in vascular perme- ability over a 64 min period in both sweet itch and normal animals. Maximal

120 A.P. Foster et al. / Veterinary Immunology and Immunopathology 44 (I 995) II_% 128

increases in vascular permeability were obtained at 32 min in each group over the dose range of PAF studied and are shown in Fig. 1 (a), The responses in sweet itch and normal animals were similar. In contrast to PAF, lyso-PAF ( 1 pg per site) did not significantly increase vascular permeability in sweet itch cases when compared with vehicle (343 + 98,44? 13 and 32 ? 5 p1.h for PAF ( 1 ,ug per site), lyso-PAF ( 1 ,ug per site) and vehicle, respectively; P< 0.05 PAF compared with vehicle, analysis of AUCs between 4 and 64 min; n = 5 ).

Histamine (0.02 and 0.2 pg free base per site), like PAF, increased cutaneous vascular permeability over a 64 min period in sweet itch and normal animals, the maximal effect occurring 32 min after i.d. injection (Fig. 1 (b) ). However, the maximal responses of sweet itch cases to histamine (0.02 ,ug per site) were signif- icantly smaller than those of normal ponies (Fig. 1 (b) ) . Histamine (0.2 pg per site) also produced smaller responses in sweet itch when compared with normal animals, although the difference was not statistically significant (P=O.O5 ) .

Culicoides antigen (50 pg per site) caused a biphasic increase in vascular permeability in sweet itch cases (Fig. 2 (a) ). The wheals were evident 15 min after injection and were maximal at 1 and 8 h. Dose-response studies carried out at the times of peak wheal formation showed that the effects of CAg were dose- related (Figs. 2 (b) and (c) )_ In normal ponies, CAg caused only a small mono- phasic increase in vascular permeability at the highest dose tested (50 ,ug per site; Fig. 2(a)).

3.2. Effects of mediators and CAg on cutaneous inflammatory cell accumulation during the active phase of the disease

3.2.1. Inflammatory cell accumulation PAF ( 1 pg per site) induced a similar degree of neutrophil accumulation at 2 h

in the dermis of sweet itch and normal animals. Maximal responses were ob- served at 2 h and very few neutrophils were present at 24 h (Fig. 3 (a) ). However, significantly greater numbers of eosinophils were evident at 2 h in affected, com- pared with normal animals (Fig. 3 (b) ). Moreover, eosinophils were still present in the skin 24 h after injection of PAF. PAF did not cause mononuclear cell num- bers to increase significantly at 2 or 24 h in sweet itch cases or normal ponies, although means were numerically greater than after injection of PBS alone in affected animals (Fig. 3 (c ) ) .

Like PAF, CAg (5 pg per site) caused neutrophil accumulation in the dermis of sweet itch cases at 2 h. However, cell numbers continued to increase up to 24 h after administration of antigen. Also CAg, like PAF, induced eosinophil accu- mulation in the skin of sweet itch cases between 2 and 24 h. Responses were maximal at 8 h. In contrast to PAF, CAg caused significant mononuclear cell infiltration in sweet itch cases at 24 h (Fig. 4 (c) ). Interestingly, there were trends towards significant positive correlations between the number of eosinophils in the skin of sweet itch cases at 8 h and the number of mononuclear cells at 24 h (r=0.79, P=O.O6) as well as increased vascular permeability at 8 h (r=0.76, P=O.O8).

A. P. Foster et al. 1 Veterinary Immunology and 1mmunopatholog.v 44 (1995) I 1 S- 128 12

-100~ I 0.0005 0.005 0.05 cl.53 5 50

CAg (pgisite)

0 0.0005 0.005 0.05 0.5 5 50

CAg (pgkite)

Fig. 2. Effect of (a) a single dose of CAg [ 50 pg per site) over a 24 h period and increasing doses of CAg at (b) I h and (c) 8 h on cutaneous vascular permeability in (m) sweet itch (n= 5) and (Cl ) normal (n=6) animals during the active phase of the disease. Each point is the mean of values ob- tained after subtraction of the vehicle response. Vertical lines represent sem. For (a), (b) and (c) P-c 0.01 when AUCs of sweet itch cases were compared with normal ponies.

In normal ponies, CAg induced only early neutrophil accumulation (Fig. 4 (a) ). Very few eosinophils or mononuclear cells migrated into the dermis of normal ponies at any time following CAg administration (Figs. 4 (b) and (c) ). Neutro- Phil, eosinophil and mononuclear cell numbers in untreated skin were very low and no different in normal and sweet itch animals (data not shown).

3.3. Effects of mediators and CAg on cutaneous vascular permeability during the inactive phase of the disease

Maximal wheal formation in response to histamine was not significantly dif- ferent during the inactive and active phases of the disease ( 36 2 6 ~1 and 13 +- 14

122 A.P. Foster et ai. 1 Veterinary Immunology and Immunopaihology 44 (1995) 115- 128

2 24 Time (h) (b) 60

2 24 Time (h)

24

Time (h)

Fig. 3. Effect ofPAF (1 fig per site) on the accumulation of (a) neutrophils, (b) eosinophils and (c) mononuclear cells in the dermis of (a) sweet itch (n = 5 ) and ( 0 ) normal (n = 6 ) animals over a 24 h period during the active phase of the disease. Each bar represents the mean of values obtained after subtraction of the vehicle response. Vertical lines represent sem. ** = PC 0.01 when compared with responses in normal ponies.

,/.d for 0.02 pg per site histamine during the inactive and active phases, respec- tively; 268 ? 135 ~1 and 22 1% 8 1 ~1 for 0.2 ,ug per site histamine during the inac- tive and active phases, respectively; n= 4). The response to the lower dose of histamine remained significantly reduced during the inactive phase when com- pared with control ponies. During the inactive phase of the disease the wheal volumes formed in response to CAg were not significantly greater than those ob- tained in response to PBS alone (Table 1) .

3.4. Effects of mediators and CAg on cutaneous inflammatory cell accumulation during the inactive season

PAF ( 1 pg per site)-induced neutrophil accumulation at 2 h was reduced dur- ing the inactive compared with the active phase of the disease,_but the difference

A.P. Foster et al. / Veterinary Immunology and Imrnunopathology 44 (I 995) I IS- 128 123

(a) 150

“E E 1 T

2

Tim”, (h) 24

(b) 200

z 1 II* n meet “Ch

T l * q normal

2 ‘de (h)

24

Fig. 4. Effect of CAg (5 pg per site) on the accumulation of (a) neutrophils. (b) eosinophils and (c) mononuclear cells in the dermis of ( n ) sweet itch (n = 5 ) and ( 0 ) normal (n = 6) animals over a 24 h period during the active phase of the disease. Each bar represents the mean of values obtained after subtraction of the vehicle response. Vertical lines represent sem. * = PC 0.05: ** = PC 0.0 I when com- pared with normal ponies.

was not significant (69 2 22 and 226 5 112 neutrophils per 4 mm2 dermis for in- active and active seasons, respectively; n=4). PAF did not cause eosinophil ac- cumulation during the inactive phase (Ok 0 and 11 t 13 eosinophils per 4 mm2 dermis; inactive and active phases, respectively; n = 4). In contrast, eosinophil as well as neutrophil accumulation in response to CAg was similar at 2 h in sweet itch cases during the inactive and active phases of the disease. However, neutro- phils, eosinophils and mononuclear cells were not evident in the skin 24 h after administration of CAg during the inactive phase (Table 2 ) .

124 A. P. Foster et al. / Veterinary Immunology and Immunopathology 44 (I 995) 1 IS- 128

Table 1 Effect of CAg (0.0005-5 pg per site) on cutaneous vascular permeability in sweet itch cases during the inactive and active phases of the disease

CAg (pg per site) Wheal volume (~1)

1 h after injection 8 h after injection

Inactive Active Inactive Active

0.0005 17+ 16 13+-l -3+ 14 818 0.005 28+8 7f4 -3k5 7k7 0.05 20+ 10 to+ 14 -I+6 175 13 0.5 926 73k22 IlflO 54+ 13 5 54222 122k 17 17-tl8 132144

Results are expressed as means I!Z sem after subtraction of the vehicle response (n = 4). Wheal volume responsestoPBSalonewere16k7~land 18klOftlat I hand lOk8~land5&5~lat8hduringthe inactive and active phases, respectively.

Table 2 A comparison of leucocyte numbers in the dermis 2 and 24 h after injection of CAg (5 fig per site) in sweet itch cases during the inactive and active phases of the disease

Leucocytes per 4 mm* of dermis

2 h after injection 24 h after injection

Inactive Active Inactive Active

Neutrophils 3Ok 10 32k20 725* 7Okl7 Eosinophils 64234 63k18 9+3* 136+24 Mononuclear cells 2k3 2+2 3+2* 44kll

Results are expressed as means k sem after subtraction of the vehicle response (n = 4). * = PC 0.05 com- pared with active phase of the disease.

Table 3 Peripheral blood leucocyte counts in sweet itch and normal animals during the active phase of the disease

Peripheral blood leucocytes ( x I 09/1 )

Sweet itch Normal

Total leucocytes 9.6*0.4* 7.3kO.7 Neutrophils 4.2kO.3 3.4kO.4 Eosinophils 0.4 * 0. I 0.3~0.1 Basophils 0.1 kO.03 0.04 f 0.02 Lymphocytes 4.4 * 0.3** 3.4 k 0.4 Monocytes 0.4to.1* 0.1 i 0.1

Results are expressed as means k sem for sweet itch (n = 5 ) and normal (n = 6) animals. * = P< 0.05; **=P< 0.01 compared with normal ponies.

3.5. Peripheral blood leucocyte counts

The total peripheral blood leucocyte count was significantly higher in sweet itch compared with normal animals. The numbers of circulating lymphocytes and monocytes were elevated in the affected group, whereas neutrophil, eosinophil

A.P. Foster et al. / Veterinary Immunology and Immunopathology 44 (1995) 1 is- I28 125

and basophil numbers were not significantly different from control ponies (Table 3). There was no significant difference between the numbers of circulating leu- cocytes in sweet itch cases (n = 4) during the inactive compared with the active phase of the disease (data not shown).

4. Discussion

The present study has shown that intradermal injection of PAF leads to oed- ema formation, as well as neutrophil and eosinophil accumulation in the skin of sweet itch cases during the active phase of the disease. Mononuclear cell infiltra- tion also occurred but the mean increase in cell numbers was not statistically significant. CAg similarly caused dose related increases in vascular permeability in these animals, with maximal responses being detected at 1 and 8 h. Neutrophil, eosinophil and mononuclear cell accumulation was also observed in response to CAg.

Antigen has been shown to cause the release of PAF from the skin of atopic human patients (Michel et al., 1988a; Shalit et al., 1989). The cellular origin of the PAF has not been elucidated but possible sources include infiltrating leuco- cytes (Lee et al., 1984) or cells resident in the skin such as endothelial cells, mast cells, dermal tibroblasts and keratinocytes (Mencia-Huerta et al., 1983; Busso- lino et al., 1986; Michel et al., 1988b, 1990). In vitro studies have shown that equine leucocytes sensitised to Ascaris ~uum produce a PAF-like lipid upon stim- ulation with antigen (Wimberley et al., 1985) and purified equine eosinophils also release PAF when challenged with ionophore ( Asmis and Jorg, 1990 ). This study has shown that PAF mimics some of the effects of antigen in the skin of sweet itch horses. Hence, if PAF is released by leucocytes recruited to the lesional sites or by the cell population resident in equine skin, it could play a role in hy- persensitivity responses to Culicoides antigen.

Similar size wheals were seen after id injection of PAF in hypersensitive and normal horses. This observation agrees with reports in which wheal and flare re- sponses to PAF were found to be no different in atopic and normal human sub- jects (Henocq and Vargaftig, 1988; Sciberras et al., 199 1; Bruijnzeel-Koomen et al., 1992). In contrast to the effects of PAF, vascular permeability responses to histamine were reduced in sweet itch, when compared with normal, horses. High circulating levels of histamine have been detected in the plasma of sweet itch cases during the active phase of the disease (Reik, 1955 ). Plasma histamine lev- els were not measured in this study but, if large amounts were present in our sweet itch cases, it would not be unexpected to see decreased responsiveness of the vas- culature following i.d. challenge with the same mediator. Histamine is, of course, likely to be a mediator of pruritus in sweet itch. However, PAF has also been reported to be pruritic after injection of small volumes into human skin (Fjellner and Hagermark, 1985 ).

PAF also caused neutrophil accumulation and a significantly greater infiltra- tion of eosinophils in the dermis of sweet itch, when compared with normal, horses

126 A.P. Foster et al. / Veterinary Immunology and Immunopathology 44 (1995) 115-128

during the active phase of the disease. It seems likely that PAF-induced eosino- phi1 recruitment is selectively enhanced in sweet itch cases, since the numbers of neutrophils and eosinophils in the circulation and in the untreated dermis of sweet itch and normal animals were no different.

The in vitro chemotactic responses of eosinophils isolated from human atopic patients to mediators such as PAF have been found to be greater than those of normal subjects (Sehmi et al., 1992; Warringa et al., 1992). Furthermore, the migration of normal human eosinophils can be enhanced by the priming effects of cytokines expressed by T cells which are present in the skin of antigen chal- lenged atopic subjects (Kay et al., 199 1; Sehmi et al., 1992; Warringa et al., 1992 ). In addition, the expression of adhesion molecules on endothelial cells is upregu- lated in inflamed human skin (Groves et al., 199 1). Hence, PAF-induced in vivo migration or adhesion to endothelial cells of equine eosinophils may be greater in sweet itch cases during natural antigen challenge. Indeed, PAF did not cause accumulation of eosinophils in the dermis of sweet itch cases during the inactive phase of the disease. It therefore seems likely that PAF-induced eosinophil re- cruitment involves priming of circulating leucocytes or dermal microvascular en- dothelial cells during the active season. Moreover, since PAF is released by equine eosinophils ( Asmis and Jorg, 1990) and is a chemoattractant for equine eosino- phils in vitro (Foster et al., 1992b), the possibility of a positive feed-back loop for the self-perpetuating recruitment of eosinophils also exists.

CAg caused neither an increase in vascular permeability nor a persistent in- flammatory cell accumulation in the dermis of sweet itch cases during the inac- tive phase of the disease. This may be due in part to a reduced level of circulating sensitizing antibodies, as reported in human allergic disease in the absence of antigen challenge (Yunginger and Gleich, 1973 ), decreasing the responsiveness of inflammatory cells and subsequent production or release of chemoattractant mediators. Furthermore, exposure to natural antigen and the subsequent produc- tion of mediators may be required for persistent inflammatory cell recruitment following i.d. administration of CAg. Seasonal variations in the effects of CAg have important implications if skin testing is to be used as an aid to diagnosis for this condition. Moreover, inter- or intra-animal comparisons of responses to CAg will only be valid if carried out during either the active or the inactive phases of the disease.

In conclusion, this study has shown that PAF, like CAg, caused increased vas- cular permeability and leucocyte accumulation in the skin of sweet itch cases. Further experiments using specific receptor antagonists for PAF, both in experi- mentally induced skin lesions and under field conditions, will help to clarify the contribution of this mediator to the antigen-induced response.

Acknowledgements

We are grateful to the Home of Rest for Horses for financial support. We also thank Mrs. R.H. Thomas and the Exmoor Pony Society for providing sweet itch cases.

‘4.P. Foster et al. / Veterinary Immunology and Immunopathology 44 (I 995) I& 128 127

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