Equine Macrophages Research Articles

Effect of strenuous exercise stress on chemiluminescence response of equine alveolar macrophages

Bronchoalveolar lavage samples were collected using a fibreoptic endoscope from horses at specified times before and after single bouts of exercise. Lucigenin-dependent phagocytic chemiluminescence was used to assess the effect of exercise on the alveolar macrophage metabolic activity in response to stimulation by opsonised zymosan. A profound suppressive effect on the chemiluminescence production was present throughout the first three days after exercise. However, the cellular composition of lavage fluids was not altered by the exercise. It is suggested that strenuous exercise may jeopardize the antimicrobial function of alveolar macrophages which may lead to an increase in susceptibility to infection.

In vitro mutagenesis

The retrovirus equine infectious anemia virus (EIAV) encodes a dUTPase situated between reverse transcriptase and integrase. We have described the Inability of EIAV with a 270-bp dUTPase deletion, ΔDU EIAV, to replicate to wild-type (WT) levels in equine macrophages (D. S. Threadgill, W K. Steagall, M. T. Flaherty, F. J. Fuller, S. T. Perry, K. E. Rushlow, S. F. J. LeGrice, and S. L. Payne, J. Virol. 67, 2592-2600, 1993). Here we describe the construction of a second dUTPase-deficient virus (DUD71E) containing a single amino acid substitution in dUTPase. ΔDU and DUD71E replicate to 2% of WT levels in macrophages by 7 days postinfection, when WT EIAV is highly cytopathic. To identify the replication block(s), we analyzed DNA synthesis, integration, and transcription. DNA synthesis was normal in macrophages, with evidence of full-length viral DNA by 24 hr postinfection. The level of integrated ΔDU and DUD71E DNA appeared to be decreased 2- to 3-fold compared to WT. Steady-state levels of full-length viral transcripts were decreased over 100-fold, indicating that replication of dUTPase-deficient EIAV is blocked between vital DNA synthesis and transcription. As dUTP hydrolysis normally plays a role in preventing incorporation of uracil into newly synthesized DNA, we investigated the possibility that dUTPase-deficient EIAV DNA contains uracil. In vitro assays showed that while WT virions do not utilize dUTP, dUTPase-deficient virus and recombinant RT synthesize uracil-containing DNA. The presence of uracil in viral DNA recovered from ΔDU- and DUD71E-infected macrophages was also demonstrated. In macrophages, a virally encoded dUTPase may be necessary to prevent the incorporation of uracil into vital DNA.

Lung Clearance of Inhaled Insoluble and Soluble Particles

Lung Clearance of Inhaled Insoluble and Soluble Particles

Hemosiderin deposits in the equine small intestine.

Intestinal samples from 30 horses used in two unrelated experiments were found to have hitherto undescribed hemosiderin deposits in the intestinal villi. There were 15 horses and ponies obtained from conventional sources and 15 laboratory-reared, strongyle-free ponies with a minimum of other parasites which included Setaria sp. and Gastvophiliis sp. All animals were reared in Louisiana and were in good condition. Complete necropsies were done on all animals and all relevant tissues were examined microscopically. Findings were consistent with the experimental designs to which these animals were submitted and did not reveal a direct association with the presence of hemosiderin deposits in the intestine. Samples were obtained from duodenum, jejunum and ileum, fixed in buffered formalin, and processed routinely. Sections were stained with hematoxylin and eosin (HE), Perk’ Prussian blue for iron [3], periodic acid-Schiff (PAS) [3], and diastase-pretreated PAS [3]. Unstained sections were viewed under incident ultraviolet light for autofluorescence. Selected samples were fixed in 2% glutaraldehyde and embedded in plastic for transmission electron microscopy. Additional formalin-fixed samples were freeze-dried and gold etched for X-ray dispersion analysis of selected areas under a scanning electron microscope. The lamina propria of the tips of the intestinal villi contained variable accumulations of iron positive deposits within macrophages (fig. I). These deposits were birefringent and light brown in HE and unstained sections. They were positive for the PAS stain and diastasepretreated PAS and did not have spontaneous fluorescence under ultraviolet light. Under electron microscopic examination, these macrophages were associated closely with arteries and capillaries (fig. 2) and fragments of the cells occasionally were found within their lumens. The deposits were always within membrane-bound structures interpreted as phagolysosomes. These were heterogeneous and contained markedly electron-dense granules, crystalloid structures, and lightly electron-dense particles (fig. 3). Occasional membrane-bound granules within the same macrophages contained lamellar structures similar to so-called myeloid figures. X-ray dispersion analysis revealed an iron peak in some villi. Macrophages observed in the horses were similar to those reported for monkeys as containing “karyolytic bodies” [I]. Vacuoles present in the equine macrophages were much smaller and fewer than found in monkeys. Changes were observed in 22 duodenal, three jejunal and three ileal sections. There was no significant difference among the strongyle-free and conventionally reared animals. When changes were present in ileum orjejunum, they also were present in the duodenum. Animals with ileal changes, however, did not necessarily have changes in the jejunum. This finding is an indication that these deposits do not progress distally from the duodenum. We conclude that these deposits are hemosiderin contained within macrophages dedicated to removing a variety of particles from the areas where the deposits are found. These changes apparently have not been described in horses previously. They are similar to those reported in the literature for macaque monkeys, baboons [I], and guinea pigs [2]. They differ from those described in monkeys in that the deposits in horses do not autofluoresce under ultraviolet light, although other characteristics are very similar.

Chemiluminescence response of equine alveolar macrophages during stimulation with latex beads, or IgG-opsonized sheep red blood cells.

Isolated equine alveolar macrophages were shown to generate a luminol-dependent light response when challenged with a phagocytic stimulus. The chemiluminescent response was not detected with luminol prepared at 1.0 x 10(-5) or 1.0 x 10(-4) molar concentrations, but was readily quantitated when used at a 1.0 x 10(-3) molar concentration. Challenge of the alveolar macrophages with latex particles or with equine IgG-coated sheep red blood cells elicited the luminol-dependent light response, whereas unchallenged equine alveolar macrophages or those challenged with unopsonized erythrocytes failed to emit light above background levels. Latex-bead-challenged macrophages released 8.06 times the total amount of light as those equine alveolar macrophages challenged with equine IgG-opsonized erythrocytes. This study represents the first investigation on chemiluminescence and equine alveolar macrophages.

Surface receptors for IgG and complement on equine alveolar macrophages.

Isolated equine alveolar macrophages obtained by bronchopulmonary lavage of four live ponies demonstrated surface receptors for equine IgG, equine IgM, and complement-coated sheep red blood cells, but not equine IgM or complement-coated erythrocytes alone. In addition, demonstration of IgG receptors was found to depend on the level of erythrocyte sensitization and could not be demonstrated by red blood cell rosetting techniques at low levels of sensitization. Demonstration of receptors for equine complement by red cell rosetting techniques required the presence of both IgM antibody and serum derived (complement) components. This is the first such study of receptors on equine alveolar macrophages.