Biochemical and functional assessment of equine lymphocyte phosphodiesterases and protein kinase C.

Lymphocytes play an important role in allergic inflammation and have been implicated in the pathogenesis of equine allergic skin and respiratory disease. Targeting intracellular signalling pathways in human lymphocytes has demonstrated a role for both phosphodiesterase and protein kinase C in cell activation. The aim of this study was to measure total cyclic nucleotide hydrolysing phosphodiesterase activity and to identify the phosphodiesterase and protein kinase C isoenzymes present in equine lymphocytes. The functional significance of these isoenzymes was then investigated by examining their role in peripheral blood mononuclear cell proliferation using isoenzyme selective inhibitors. Total cyclic adenosine monophosphate hydrolysing phosphodiesterase activity was double that of cyclic guanosine monophosphate (30+/-2 pmol/min mg versus 16+/-3 pmol/min mg for cyclic adenosine and cyclic guanosine monophosphate phosphodiesterase activity, respectively). Evidence for the presence of PDE1, 3, 4 and 5 was obtained and PKCalpha, beta, delta, eta, iota, theta and zeta were identified. Selective inhibitors of PDE4, PKCdelta and conventional PKCs alpha and beta caused significant inhibition of mitogen-induced peripheral blood mononuclear cell proliferation. This study demonstrates a functional role for specific signalling isoenzymes and suggests that, in the context of allergic inflammation, targeting inflammatory cells involved in disease pathogenesis with relevant isoenzyme inhibitors may have therapeutic potential.

Role of the chemokine eotaxin in the pathogenesis of equine sweet itch.

The chemokine eotaxin is involved in the recruitment of eosinophils and T helper 2 lymphocytes in human allergic diseases, and drugs that block its activity, including eotaxin receptor (CCR3) antagonists, are being developed. The authors have recently cloned the horse ortholog of eotaxin and shown that it can induce equine eosinophil migration and activation in vitro. Moreover, eotaxin mRNA expression was upregulated in cultured horse dermal fibroblasts exposed to equine interleukin-4, suggesting a possible source of this eosinophil chemoattractant in equine skin. The results of this study show that eotaxin and monocyte chemoattractant protein (MCP) 1, but not MCP-2 or MCP-4, mRNA expression is upregulated in skin biopsies of sweet itch lesions when eosinophils are present, when compared with clinically normal skin from the same ponies.

T cell lines generated with type II collagen proliferate in an autologous mixed lymphocyte response.

Collagen-induced arthritis (CIA) is a T cell-dependent disease induced in susceptible rodents by immunizing with bovine type II collagen (bCII). In order to study T cell responses, a programme to generate bCII-specific T cell lines from arthritic rats was initiated. Lymph node cells from bCII-immune WA/KIR/kcl rats were cultured with bCII in vitro, and the T cells were isolated and restimulated with bCII-pulsed antigen presenting cells (APC) (thymus cells or splenic low density cells). However, T cells, generated initially to bCII, subsequently proliferated upon co-culture with syngeneic APC even in the absence of bCII. This suggests that exposure to bCII resulted in the activation of a population of self-reactive T cells which proliferate in an autologous mixed lymphocyte response. In contrast, short-term T cell lines generated to ovalbumin, heat-denatured bCII and the collagen peptide bCII(184-198) proliferated in response to specific antigen-pulsed APC without demonstrating self-reactivity. Since denatured bCII and bCII(184-198) peptide are not arthritogenic and failed to generate self reactivity in vitro, this suggests that the native triple helical conformation of bCII is required for stimulating autoreactive T cell responses.

Antigen presentation of Type II collagen in rats.

Collagen-induced arthritis (CIA) is a T-cell dependent disease of rats which follows immunization with bovine type II collagen (bCII). Susceptibility to CIA is linked to the genes encoding the major histocompatibility complex (MHC), suggesting that antigen presentation is important in disease pathogenesis. Antigen-presenting cells (APC) (macrophages, dendritic cells (DC) and B cells) were prepared from WA/KIR/KCL rats and presentation of antigen, in the form of native protein (bCII) or synthetic peptide (bCII:184-198), was assessed in T-cell proliferation assays. Whilst macrophages inhibited proliferative responses to bCII, splenic or thymic low density cells, enriched for DC, presented both bCII and bCII(184-198) peptide. However, bone marrow-derived DC, which stimulated T-cell responses to OVA, failed to present bCII, suggesting differences in processing of these two antigens. B-cell depletion from lymph node cells abrogated the proliferative response to bCII and reconstitution of a T-cell population with B cells restored the proliferative response, indicating that B cells are important for stimulating T-cell responses to bCII. B cells play a critical role in CIA by producing pathogenic anti-bCII antibodies, and we propose that B cells are also important APC which present bCII to CD4+ T cells.

The anti-human CD21 antibody, BU33, identifies equine B cells.

The number of antibodies for identifying equine B cells is small and the number that react with well-defined epitopes even smaller. The monoclonal antibody, BU33, which is directed against human CD21 (Complement Receptor 2; CR2) was shown to identify (1) follicular lymphocytes in the lymph nodes and spleen of three horses, and (2) a mean of 18 +/- 6% (SEM) of peripheral blood lymphocytes from 10 horses. These findings indicate that the antibody identifies equine B cells and possibly equine CR2 or a related molecule. This study adds to the reagents available for equine research and diagnostic pathology.

Culicoides antigen extract stimulates equine blood mononuclear (BMN) cell proliferation and the release of eosinophil adherence-inducing factor(s).

Intradermal injection of a Culicoides antigen extract (CAgX) induces T lymphocyte and eosinophil accumulation in the skin of horses with sweet itch. Blood mononuclear (BMN) cells from normal ponies proliferate when stimulated by mitogen (phytohaemagglutinin, PHA) or antigen (tetanus toxoid, TT) and, as shown here, release soluble factor(s) that induce eosinophil adherence. CAgX also caused concentration dependent proliferation of BMN cells from sweet itch and normal ponies [stimulation index: 29 (13) and 17 (7) for BMN cells from sweet itch and normal ponies, respectively during the active phase of disease; 4 microg protein ml(-1)CAgX; 168 h]. A heat labile factor(s) which caused eosinophil adherence was also released [sweet itch ponies: 6.0 (1.6) per cent adherence versus 1.3 (0.4) per cent; normal ponies: 6.6 (0.5) per cent adherence versus 0.9 (0.1) per cent for supernatants from CAgX (4 microg protein ml(-1); 48 hours) stimulated versus unstimulated BMN cells, respectively]. These results suggest that soluble proteins released from T lymphocytes could affect eosinophil function in the lesional skin of sweet itch horses.

Infection of endothelial cells with equine herpesvirus-1 (EHV-1) occurs where there is activation of putative adhesion molecules: a mechanism for transfer of virus.

Evidence is presented to show that activation of endothelial and leucoyte adhesion molecules is a key step in transferring virus from infected leucocytes; and determines the restricted tissue tropism. A range of tissues from 2 experimentally infected mares in late pregnancy at 4 and 8 days after infection with EHV-1 were compared with those from normal pregnant and nonpregnant mares. Rabbit antisera to equine activated endothelial cell molecules were used to identify which tissues expressed these molecules in normal nongravid and gravid mares, and to investigate whether the range of tissues was altered in pregnant mares as a consequence of infection. The results indicated that the endothelium of the pregnant reproductive tract did express these molecules. In the 2 pregnant mares infected with EHV-1, the endothelial cells in the nasal mucosa also expressed these activated endothelial cell molecules. Therefore, the sites expressing these molecules closely correlated with those where virus infection of endothelial cells has been described and is consistent with experimental in vitro data, indicating that expression of these molecules is an essential stage in the transference of virus from leucocytes to endothelial cells.

Cloning of equine chemokines eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2 and MCP-4, mRNA expression in tissues and induction by IL-4 in dermal fibroblasts.

We report the cloning of four equine CC chemokines, eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2 and MCP-4, which show high levels of identity with their respective homologous sequences in other species. Using a multiplex RT-PCR, we have studied the constitutive mRNA expression of these four CC chemokines in skin, lung, liver, spleen, jejunum, colon and kidney of normal adult horses and compared this data with the eosinophil counts in the same samples. We demonstrate that eotaxin mRNA is only expressed in jejunum and colon, where there are large numbers of eosinophils suggesting that eotaxin might be recruiting eosinophils in the normal digestive tract of the horse. MCP-1 and MCP-4 are expressed in all tissues whereas MCP-2 is only found in some samples of lung, spleen, liver and kidney. We also report the early induction (2h) of equine eotaxin and MCP-4, and the up-regulation of MCP-1 by interleukin-4 in dermal fibroblasts, suggesting these chemokines might be involved in equine skin allergic diseases.

Equine macrophage identification with an antibody (Ki-M6) to human CD68 and a new monoclonal antibody (JB10).

Monoclonal antibodies (mAbs) recognizing equine macrophages are scarce. The present study compared the immunocytochemical staining of various equine tissues (lymphoid tissue, lung, liver, small intestine, skin and blood leucocytes) by an antibody, Ki-M6, which detects CD68 in human macrophages and dendritic cells, and by a new anti-equine mAb, JB10, with staining produced by two previously described anti-equine macrophage mAbs, CZ2.2 and CZ3.3. Ki-M6 was shown to identify equine macrophages, which had a distribution different from those identified by CZ2.2 and CZ3.3. JB10 identified equine macrophages with a distribution similar to those identified by Ki-M6, but additionally bound to polymorphonuclear leucocytes. Flow cytometry of peripheral blood leucocyte subpopulations and tissue immunocytochemistry were used to compare staining by JB10 with that of CZ2.2 and CVS19; the latter identifies the myeloid antigen, EqCD13, found on polymorphonuclear leucocytes. The staining by JB10 differed from that of both CZ2.2 and CVS19, suggesting that JB10 detects a different molecule. These additional mAbs should prove useful for the future study of new, defined, populations of macrophages in equine immune responses and pathology, and, in the case of Ki-M6 antibody, may make possible an analysis of the structure, distribution and function of the CD68 molecule in the horse.

Characterisation of lymphocyte subpopulations in the skin and circulation of horses with sweet itch (Culicoides hypersensitivity).

Circulating lymphocyte numbers are elevated in horses with the allergic skin disease sweet itch and skin lesions are typified by an infiltrate of eosinophils and mononuclear cells, the latter of which have not been fully characterised. The aim of the present study was to characterise the lymphocyte subpopulations in the circulation and skin of ponies with sweet itch by flow cytometry and a newly developed modified alkaline phosphatase immunohistochemical technique. Sweet itch ponies were found to have significantly greater numbers of circulating CD5+ and CD4+ T-lymphocytes than normal animals. Increased numbers of CD3+ T-lymphocytes, most of which were CD4+, and eosinophils were present in the skin of these animals following intradermal injection of a Culicoides antigen extract (97 +/- 21 vs. 449 +/- 49 CD3+ T-lymphocytes/mm2 in deep dermis of vehicle vs. antigen injected sites; 83 +/- 8% CD4+ T-lymphocytes at antigen injected site). T-lymphocytes, which are thought to be important in the pathogenesis of human allergic skin disease, may therefore contribute to the development of sweet itch lesions via the release of cytokines which can cause eosinophil accumulation and activation. An understanding of the pathology of this disease may lead to a more rational approach to therapy.

Mammalian granulocyte-macrophage colony-stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in fish.

A eukaryotic plasmid DNA carrying the AACGTT CpG motif in its ampR gene is a 'danger' signal for mice and caused an increase in the specific antibody titres of fish and mice after immunization with beta-galactosidase (beta-gal). A second pUC-based plasmid, which is inactive in mice and contains the GACGTC CpG motif in its cytomegalovirus (CMV) promoter, had no effect on antibody responses to beta-gal in either fish or mice. A synthetic oligonucleotide, which contains the GACGTT motif, potentiated antibody responses to co-administered beta-gal protein in mice, but not in fish. This is early evidence that lower and higher vertebrates recognize different unmethylated CpG motifs as 'danger' signals. In addition, plasmid DNA expressing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) had a marked effect on cytotoxic T-cell-like activity in fish by reducing the average number of myofibres that expressed beta-gal, 28 days after co-injection with plasmid DNA expressing beta-gal. Although the mechanism by which the mouse GM-CSF exerted its biological effects in fish is unknown, this finding might have important implications for fish vaccination, particularly when cytotoxic T cells may play a critical role.

Epithelial lymphocyte and macrophage distribution in the adult and fetal equine lung.

Leucocytes in the lung epithelium play an important role in the ability of an animal to respond appropriately to inhaled pathogens. The distribution of lymphoid and myeloid cells associated with the lung epithelium was examined immunohistochemically throughout the respiratory tract of four horses, comprising two adults from an abattoir, one pregnant mare, and her fetus (in the final stage of gestation). Cross and tangential cryosections were labelled with monoclonal antibodies against T-cell, B-cell, macrophage/dendritic myeloid cell, and major histocompatibility Class (MHC) II surface antigens. Cell numbers were determined by microscopy. In the three adult horses, epithelial CD3+ T-cell numbers decreased progressively from the upper to the lower respiratory tract, but in the fetus there were low numbers of T cells (at most, 10% of those seen in the adult airways) and little variation in different parts of the respiratory tract. MHC Class II was expressed on the airway epithelium of the two abattoir horses, but not that of the mare and her fetus. In these two animals occasional large, mostly irregularly-shaped, Class II-positive cells were seen. Very few epithelium-associated cells in any animal were labelled by anti-CD21 antibody, which identifies B cells, or anti-myeloid cell antibodies; an anti-rat macrophage antibody (ED2) was shown, for the first time, to identify mature equine alveolar macrophages. Despite the small number of animals, the results suggest that in normal adult horses the greatest numbers of epithelial T cells are found where there is greatest contact with airborne antigens, and that there is constitutive epithelial MHC Class II expression. The low level of MHC Class II expression in the fetus, together with the reduced numbers of T cells, was consistent with the suggestion that the fetal immune system requires exposure to airborne stimuli for full development. The low level of MHC Class II expression in the mare may have reflected the immunosuppression that accompanies pregnancy.

In vitro reactivation of latent equid herpesvirus-1 from CD5+/CD8+ leukocytes indirectly by IL-2 or chorionic gonadotrophin.

IL-2 and equine chorionic gonadotrophin (eCG) initiated reactivation of equid herpesvirus-1 (EHV-1) from venous lymphocytes at a frequency of 1/10(-5). Indirect immunofluorescence showed that > 80% of virus-positive leukocytes were CD5+/CD8+ with the remaining 20% being CD5+/CD8-/CD4-. Cocultivation demonstrated that the reactivated virus was infectious. In addition, virus was reactivated in vitro from leukocytes of > 70% of horses by the mitogens phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Transfer of supernatants showed that IL-2 and eCG acted indirectly by causing the release of other mediators from adherent cells; these mediators then reactivated EHV-1 from T cells. Blocking experiments with anti-IL-2 showed that PWM and PHA acted via IL-2 but that eCG did not. This is the first clear definition of the lymphoid cells that harbour latent EHV-1 in vivo and correlates with current RT-PCR and in situ hybridization of latency-associated transcripts in lymphocytes. This method of reactivation in vitro can be used to detect horses carrying latent EHV-1 in vivo and also has the potential to dissect the sequence of events involved in reactivation in vitro.

Quantitative and qualitative immunohistochemistry of T cell subsets and MHC class II expression in the canine small intestine.

In man and rodents, cells of the gastrointestinal immune system include B and T lymphocytes, granulocytes, macrophages and dendritic cells. Abnormalities in leucocyte numbers and function have been described in diseases of humans, such as coeliac disease and inflammatory bowel disease. The purpose of this study is to describe the normal distribution of T cells and MHC Class II expression in the small intestine of clinically normal dogs, to allow subsequent comparison with disease states. Full-thickness sections of duodenum, jejunum and ileum from seven young adult beagle dogs were immediately snap-frozen following euthanasia. Avidin-biotin-enhanced immunocytochemistry was used to detect expression of canine CD3, CD4, CD8 and MHC Class II antigens. Positively stained lamina propria cells were quantified using an eyepiece graticule, and positively stained intraepithelial cells by counts per 100 epithelial cells. In the lamina propria, the density of all leucocyte subsets was significantly increased towards the villus tip for all regions (p < 0.05). There was no apparent difference in the distribution of CD3+, CD4+ and CD8+ leucocytes between the three portions of the small intestine. The ratios of CD4+ cells to CD8+ cells in the lamina propria and epithelium were 59:41 and 15:85, respectively. Subtractive analysis suggested that 50-55% of CD3+ intraepithelial cells were CD4-CD8-. MHC Class II expression was apparent upon lamina propria cells with a dendritic morphology, as well as round cells. Epithelial MHC Class II expression was apparent in 7/7 ileal sections, compared with only 1/7 duodenal and 1/7 jejunal sections. This study shows that the small intestinal mucosa of the dog contains similar leucocyte populations to those found in other species, and suggests that these cells may play similar roles in gastrointestinal immunity.

Adhesion molecule expression on circulating lymphocytes and monocytes in patients with isolated idiopathic retinal vasculitis.

Idiopathic retinal vasculitis (RV) is a disease of unknown aetiology in which immune responses are involved in the pathogenesis of disease. T cells are thought to be important in this disease and there is evidence of peripheral T cell activation in a significant proportion of patients. The authors examined the expression of the leukocyte adhesion molecules (LeuCAMs) CD11a and CD18 on the peripheral T cells and monocytes of 11 patients with active idiopathic retinal vasculitis compared with 11 age, sex and race matched controls. Although the percentage of T cells expressing HLA DR was increased in the patient group the percentage of cells expressing CD11a and CD18 and the density, expressed as mean fluorescence intensity (MFI) were no different in the two groups. The expression of CD11a and CD18 on peripheral blood monocytes was also not found to be different between patients and controls. Adhesion between leukocytes and endothelial cells is essential for emigration of leukocytes and their accumulation in disease. Our findings suggest that any upregulation of leukocyte adhesion molecules occurring as part of this process is taking place in response to locally produced cytokines.

Altered expression of CD11/CD18 on the peripheral blood phagocytes of patients with tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is characterized by granulomatous lesions made up of epithelioid cells, giant cells and mononuclear leucocytes. Cell-cell adhesion is important in granuloma formation and in the leucocyte migration which accompanies it. We have recently shown increased expression of the adhesion molecules CD11/CD18 (LeuCAMs, beta 2 integrins) on peripheral blood leucocytes from patients with sarcoidosis (Shakoor & Hamblin, 1992). Here we have studied the expression of CD11/CD18 and CD29 (VLA beta 1 integrin) on the peripheral blood leucocytes of 10 TB patients by flow cytometry. The density (expressed as mean fluorescence intensity) of CD11b on monocytes and polymorphs was increased (P < 0.005), as was CD11c (P < 0.005) and CD18 (P < 0.05) on polymorphs. CD11a expression was significantly reduced on polymorphs (P < 0.05). No differences were found in the expression of CD29, the percentages of cells expressing any molecule and, in contrast to sarcoidosis, the density of any molecule on lymphocytes. Although the cytokine tumour necrosis factor (TNF) has been implicated in the process of up-regulation, an ELISA for TNF failed to detect significant levels in plasma. The results suggest increased peripheral phagocyte CD11/CD18 expression is a feature of TB, which may contribute to the pathological processes involved.

Antigen-presenting capacity in normal human dermis is mainly subserved by CD1a+ cells.

A proposed role for antigen-presenting dermal dendrocytes in the pathogenesis of many dermal inflammatory skin diseases remains speculative. We therefore sought to determine the phenotype and functional characteristics of antigen-presenting cells isolated from normal human dermis. Normal adult human skin was incubated overnight with dispase at 4 degrees C, the epidermis was removed, and the residual dermal preparation was then minced and digested with a mixture of hyaluronidase, collagenase, and DNAase at 37 degrees C, prior to filtration through mesh. Dermal cell suspensions thus obtained were stained using specific monoclonal antibodies, and analysed by fluorescence microscopy or flow cytometry. Mean values were as follows: CD45+ leucocytes 39%, HLA-DR+ cells 39%, Ulex europaeus agglutinin I+ endothelial cells 26%, CD1a+ cells 3.9%, CD11b+ cells 16%, CD11c+ cells 6%. Mitomycin C-treated crude dermal cell suspensions induced allostimulation of peripheral blood mononuclear cells in a 7-day culture, as assessed by 3H-TdR incorporation. Depletion of CD1a+ Langerhans-like cells from the dermal cell preparation, by 95, 74 and 90% in three separate experiments using immunomagnetic beads, reduced 3H-TdR incorporation at optimal responder-to-stimulator cell ratios by 90, 64, and 87%, respectively. Our findings suggest that, in normal human dermis, the great majority of the alloantigen-presenting capacity resides in the CD1a+ Langerhans cell-like dendritic antigen-presenting cell population, and not to any great extent in either CD1a- macrophage-like cells, or HLA-DR+ endothelial cells. The relationship of the CD1a+ dermal antigen-presenting cells to the Langerhans cell lineage remains to be determined.

Circulating adhesion molecules in sarcoidosis.

Sarcoidosis is a disease of unknown etiology characterized by non-caseating granulomata together with a number of systemic abnormalities. We have recently shown these include increased expression of the integrins CD11/CD18 on peripheral blood leucocytes. Here we have measured serum levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1) in 23 patients and 14 normal controls using antigen capture sandwich ELISAs. Median circulating E-selectin levels in the patients were nearly three times those of the controls (P < 0.0001, Mann-Whitney U-test), whilst ICAM-1 but not VCAM-1 levels were only slightly elevated. These results show that endothelial cell activation and shedding of E-selectin into the circulation are additional features of the pathology of sarcoidosis.

Increased CD11/CD18 expression on the peripheral blood leucocytes of patients with HIV disease: relationship to disease severity.

In HIV disease increased adhesion between leucocytes themselves and between leucocytes and endothelium may contribute to cell loss and viral spread. Using a novel method for the preparation of blood leucocytes for flow cytometry, we report increased expression of leucocyte adhesion molecules (LeuCAMs) (CD11/CD18) on peripheral blood leucocytes of patients with HIV disease compared with normal controls. Patients were divided into two groups on the basis of CD4 T lymphocyte numbers (those with > 0.5 x 10(9)/l and those with < 0.2 x 10(9)/l), and assessed for p24 antigen expression, viral load and serum tumour necrosis factor (TNF) levels as well as LeuCAM expression. Patients with < 0.2 x 10(9)/lCD4 cells had more p24 antigen and more HIV infectious virus and more serum TNF than those with > 0.5 x 10(9)/l. Whilst the percentages of only monocytes and polymorphs expressing CD11b were significantly increased in patients with the least CD4 cells, the density of LeuCAMs, expressed as mean fluorescence intensity (MFI), was significantly increased on all leucocytes, with the most significant increases being seen on patients with the fewest CD4 T cells. Our findings are consistent with leucocyte activation by a soluble factor, although we could find no correlation between levels of TNF and LeuCAM expression. The increased expression of adhesion molecules on peripheral blood leucocytes could play a role in the cellular extravasation and aggregation seen in HIV disease.