Abstract
Plasma histamine levels were measured in 11 clinically healthy cats and 15 cats with allergic dermatitis. Histamine levels were markedly elevated in 5/15 allergic cats. A calcium ionophore, A23187, stimulates histamine release from feline peripheral blood cells. Immunostaining of blood smears from clinically healthy cats revealed that approximately 10% of eosinophils possessed histamine-containing granules. These results indicate that some peripheral eosinophils in cats contain histamine and can release histamine by appropriate stimulation.
Histamine is an important chemical mediator of allergic reactions in humans (Meyer et al 1983). In its inactive form, histamine is stored mainly in mast cells and basophils (Wasserman 1983). Stimulation of sensitised allergens or calcium ionophores can induce histamine release from these cells (Lichtenstein and Osler 1966, Meyer et al 1983, Wasserman 1983, Beer et al 1984, Yamashita et al 2000). Histamine binds mainly to the H1 receptor expressed on blood vessels and smooth muscle tissues. Thereby, it provokes type-I allergic reactions including vasodilation, smooth muscle arctation (Douglas 1975), and stomach acid secretion (Sandvik et al 1987). These reactions are associated with clinical signs of various allergic conditions such as atopic dermatitis, allergic asthma, and anaphylaxis (Bruce et al 1976, Hammerberg, et al 2001). Elevation of histamine levels in serum and tissues has been demonstrated in human patients with allergic conditions (Sugiura et al 1984).
Cats are also affected with various allergic conditions, including atopic dermatitis, food hypersensitivity, flea-allergy dermatitis, and allergic asthma. Immunological and histopathological findings in allergic cats resemble those in human patients: focal accumulations of eosinophils, marked eosinophilia, and increased levels of serum antigen-specific IgE (Norris et al 2003, Roosje et al 2004). Histamine H1 receptor blockers have been used to control allergic conditions in cats (Norris et al 2003). It has been speculated that histamine is a major chemical mediator of type-I allergic reactions in cats. This study is intended to measure histamine levels in normal cats and cats with allergic dermatitis. Histamine releasing cells in feline peripheral blood are also investigated.
Materials and Methods
Animals and Blood Collection
Peripheral blood samples were collected from 11 clinically healthy cats and 15 cats with allergic dermatitis. These 15 cats were admitted to the University Veterinary Clinic at Tokyo University of Agriculture and Technology for diagnosis and treatment. Of the 15 cats, 14 were diagnosed as having atopic dermatitis by clinical signs and by exclusion of infectious and parasitic diseases according to criteria applied by Roosje et al (2004). One cat was diagnosed as having a food allergy based on an antigen-specific IgE test and improvement of clinical signs by a hypoallergic diet. Peripheral blood samples were collected by venepuncture. Sera were sent to commercial laboratory (Hitachi Ltd, Japan) for detection of antigen-specific IgE antibody using human recombinant high affinity IgE receptor α-chain (FcεR1α; Wasson and Grieve 1998). Complete blood counts and differential white blood counts were measured in 10 of the 15 pruritic cats (Table 1).
Signalment and clinical findings in 15 cats with allergic dermatitis
Sensitised antigens were determined by antigen-specific IgE test.
F = female.
NF = neutered female.
M = male.
NM = neutered male.
ND = not done.
HDM = house dust mite.
Cell Stimulation with Calcium Ionophore A23187
The calcium ionophore A23187 (Sigma–Aldrich Corp., St Louis, MO) was used as a non-immunological stimulus for histamine release. The reagent was resolved in dimethyl sulfoxide. The calcium ionophore was added to 1 ml heparinised peripheral blood at the final concentration of 5 μM. The peripheral blood sample was incubated at 37°C for 30 min to induce histamine release. After centrifugation for 10 min at 300 g, a plasma sample was collected for histamine measurement.
Histamine Assay
Histamine levels in plasma were determined using an enzyme-immunosorbent histamine assay kit (Histamine Enzyme Immunoassay Kit; SPI-Bio, Massy Cedex, France) according to the manufacturer's instructions. Briefly, histamine-acetylcholinesterase (AChE) tracer and the plasma sample were incubated in wells that were pre-coated with a mouse anti-histamine antibody. The plate was washed to remove unbound reagents. Ellman's Reagent (enzymatic substrate for AChE and chromogen) was added to the wells. The AChE tracer acts on Ellman's Reagent to form a yellow compound. The optical density at 405 nm was determined using a spectrophotometer.
Histamine Immunostaining
Immunostaining against histamine was performed to identify histamine-possessing cells in feline peripheral blood. Peripheral blood samples were collected from three clinically healthy cats. A feline mast cell sample taken from an enlarged lymph node of a cat with visceral mast cell tumour was also used as a positive control. Blood and biopsy samples were smeared on slide glasses and fixed in 4% 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Sigma–Aldrich Corp.) in PBS at room temperature for 2 h (Jacobi et al 2000). After washing three times with PBS, endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxidase in methanol for 20 min at 4°C. After washing, non-specific binding was blocked by incubation with 3% skim milk (Meiji Dairies Corp., Tokyo, Japan) followed by further incubation with 10% normal goat serum for 20 min. These samples were washed and incubated with rabbit antiserum to histamine (Chemicon Co. Ltd, Temecula, CA) or rabbit serum as a negative control diluted in phosphate buffered saline (PBS) (1:200) overnight at 4°C. After washing with PBS, the slides were incubated for 5 min with peroxidase-conjugated goat anti-rabbit IgG (AffiniPure F (ab′) fragment: Jackson Immuno Research Laboratories, Inc., West Grove, PA) at room temperature. After a final wash, the slides were reacted with 3,3′-diaminobenzidine (DAB) substrate chromogen (Dako Envision kit; DakoCytomation Co. Ltd, Carpinteria, CA) for 20 s. The slides were counterstained with haematoxylin and eosin for double staining.
Results
Plasma Histamine Level
Plasma histamine levels were determined in 11 clinically healthy cats and 15 cats with allergic dermatitis (Fig 1). The histamine levels of the 11 clinically healthy cats were 44.7±23.9 (mean±SD) nM, ranging 14.6–89.0 nM; those of 15 cats with allergic dermatitis were 84.7±62.5 nM ranging 18.4–206.8 nM. Histamine levels were markedly elevated in five of the 15 allergic cats beyond 92.5 nM (mean+2 SD of 11 clinically healthy cats). No correlation was found between the plasma histamine level and the number of peripheral blood eosinophils (r2=0.0493) or basophils.
Plasma histamine levels of 11 clinically healthy and 15 cats with allergic dermatitis were determined using ELISA.
Histamine Release from White Blood Cells by A23187 Stimulation
Peripheral blood samples from six clinically healthy cats were stimulated with A23187 to investigate whether peripheral blood cells can be a source of plasma histamine. Plasma histamine levels were evaluated using enzyme-linked immunosorbent assay (ELISA) (Fig 2). The respective plasma histamine levels before and after stimulation were 27.2±25.7 (mean±SD) and 91.2±65.3. Plasma histamine levels were markedly elevated in three of the six cats after stimulation. No correlation was apparent between histamine levels after stimulation and the number of eosinophils (Table 2, r2=0.2731). These results indicate that peripheral white blood cells in cats can release histamines when appropriate stimuli were applied to them.
Histamine release with calcium ionophore stimulation
Peripheral blood samples were collected from six clinically healthy cats. Plasma histamine levels were measured before and after calcium ionophore (A23187) stimulation.
Identification of Histamine-containing Cells in Feline Peripheral Blood
Blood-smear samples were prepared to identify the histamine-containing cells in peripheral blood. As a positive control, mast cells from a cat with mast cell tumour were stained using the same procedure. Histamine-containing granules were detected in cytoplasm of the mast cells (Fig 3A). These mast cells were not stained when rabbit serum was applied as the primary antibody instead of the antiserum to histamine (Fig 3B). In blood-smear samples from a healthy cat, histamine was detected in granules of some polynuclear cells (Fig 3C). Haematoxylin and eosin staining revealed that histamine-containing cells were eosinophils. Approximately 10% of eosinophils possessed histamine-containing granules (Fig 3E1). Neutrophils (Fig 3N), lymphocytes and a most eosinophils (Fig 3E2) were negative for the staining. Similar results were obtained using blood-smear samples from the other two healthy cats (data not shown).
(A) Mast cells from a cat with mast cell tumour as a positive control. A smear sample was stained with anti-histamine antibody (original magnification ×1000). (B) Fine needle aspiration was smear-stained with rabbit serum as a negative control (original magnification ×1000). (C) Peripheral blood smear obtained from a clinically healthy cat. A smear sample was stained with anti-histamine antibody (original magnification ×400). The same smear slide was stained with haematoxylin and eosin. (E1), (E2), and (N) are identical to the cells shown in panel (C). (E1): Histamine positive eosinophil. (E2): Histamine negative eosinophil. (N): Histamine negative neutrophil (original magnification ×400).
Discussion
The present study revealed that plasma histamine levels were markedly elevated in five of the 15 cats with allergic dermatitis. Similar findings have been reported that plasma histamine levels were elevated in some human patients with atopic dermatitis (Ring et al 1979, Koshibu 1994, Kimura et al 1999, Imaizumi et al 2003). The elevation of plasma histamine levels has been demonstrated by a food challenge test in patients with food allergy (Sampson and Jolie 1984, Koshibu 1994). Although the origin of plasma histamine has not been determined, it has been suggested that plasma histamine was released from infiltrating inflammatory cells in human patients with atopic dermatitis (Hanifin et al 1985). The present study revealed that plasma histamine levels were markedly elevated in five of the 15 cats with allergic dermatitis. This result implies that histamine could play a role in allergic conditions in some cats, just as it does in humans (Metcalfe et al 1997). However, the reasons for the differences in plasma histamine levels among allergic cats are unknown. We speculate that plasma histamine levels might represent release from cells localised in affected organs in acute allergic conditions, as seen with the food challenge test in human allergic patients.
Our results indicate that some leukocytes in feline peripheral blood possess histamine and can release histamine by calcium ionophore stimulation. Histamine release from peripheral blood leukocytes has been reported in human allergic patients (Butler et al 1983, Wahn et al 1990). Basophils are responsible for the histamine release in humans; however, they are known to be rare in feline peripheral blood. In this study, immunostaining for histamine revealed that histamine was present in the cytoplasmic granules of some eosinophils in feline peripheral blood. Approximately 10% of eosinophils in feline peripheral blood were positive to histamine staining. Human blood eosinophils are classifiable into subpopulations according to their specific gravity (Shult et al 1988, Kloprogge et al 1989). These eosinophil subsets are considered to represent different stages of activation by IL-3, IL-5 and GM-CSF (Shult et al 1988, Kloprogge et al 1989). In humans, the expression of the low-affinity IgE receptor (FcεRII) has been identified on the surface of hypodense eosinophils; members of this subset of eosinophils release eosinophil peroxidase (EPO) and a major basic protein (MBP) when they combine with IgE (Shult et al 1988, Kloprogge et al 1989). Butler et al (1983) reported that maximal histamine release from leukocytes in humans was not correlated with the number of basophils or the histamine content per basophil, but with cyclic AMP phosphodiesterase activity in leukocytes. They hypothesised that enhanced histamine releasability from leukocytes might be related to an abnormality in cyclic nucleotide regulation. We assumed that feline eosinophils could release histamine at a specific stage of activation, and the difference in histamine release among individuals may reflect the histamine releasability of eosinophils.
Eosinophils are an important indicator of allergic dermatitis in cats. One of the characteristic signs of feline allergic dermatitis is the eosinophilic granuloma complex which is characterised by prominent eosinophilic infiltrates in tissue (Roosje et al 2004). Our results suggest that eosinophils could play a role in the pathogenesis of feline allergic diseases by inducing a histamine-mediated allergic reaction. Treatments that target eosinophils might help to establish an effective therapeutic strategy for feline allergic diseases.