On-line version ISSN 1678-4162
Arq. Bras. Med. Vet. Zootec. vol.55 no.3 Belo Horizonte June 2003
Avaliação da função dos macrófagos alveolares após infecção experimental em cavalos por herpesvírus eqüino tipo 1
E. MoriI, *; C.M.C. MoriII; A.M.M.P. Della LiberaI; M.C.C.S.H. LaraIII; W.R. FernandesI
IDepartamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo
IIDepartamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo
IIICentro de Pesquisa e Desenvolvimento de Sanidade Animal, Instituto Biológico de São Paulo
The role of the pulmonary alveolar macrophages (PAM) in the lung defense mechanism was evaluated in horses infected with equine hespesvirus-1 (EHV-1). Five adult horses were exposed to 106.6 TCID50 EHV-1 by intranasal instillation. Cytology of bronchoalveolar lavage (BAL) was performed using cytocentrifugation of samples and slides stained by Rosenfeld. Cell concentration was adjusted to 2´106 cells/ml, for the measurement of macrophage activity - spreading, phagocytosis of zymosan particles and release of hydrogen peroxide (H2O2). All animals were positive in virus isolation on the second, third and fifth days post-inoculation (DPI). Seroconversion was observed on the 14th DPI. Lymphocytosis was observed by BAL cytology on the 16th DPI. Measurement of macrophage activity demonstrated a marked increase in the spreading rate, on the 23rd and 30th DPI. Phagocytosis was decreased on the second DPI, and returned to levels similar to those observed before inoculation on the 23rd DPI. The amount of H2O2 released by PAM declined on day 2, but, by day 16, they returned to values similar to those observed before inoculation. The decline in PAM activity in the acute phase of disease is indirect evidence that these cells have an important role in lung defense mechanisms against this agent.
Keywords: equine, alveolar macrophage, equine herpesvirus, EHV-1, bronchoalveolar lavage
O papel dos macrófagos alveolares (MA) nos mecanismos de defesa pulmonar foi estudado em cavalos infectados pelo herpesvírus eqüino tipo 1 (EHV-1). Cinco cavalos adultos foram inoculados com 106,6 TCID50 do EHV-1, por instilação intranasal. A citologia do lavado broncoalveolar (LBA) foi feita usando-se citocentrifugação das amostras e confecção de lâminas coradas por Rosenfeld. A concentração celular foi ajustada para 2´106 células/ml, para mensuração da atividade macrofágica - espraiamento, fagocitose de partículas de zymosan e liberação de peróxido de hidrogênio (H2O2). Observou-se soroconversão no 14o dia pós-inoculação (DPI) e isolamento viral positivo no segundo, terceiro e quinto DPI. Na citologia do LBA observou-se linfocitose no 16o DPI. Os testes de mensuração da atividade macrofágica demonstraram aumento significativo nos índices de espraiamento no 23o e 30o DPI e diminuição no índice de fagocitose no segundo DPI, retornando a valores semelhantes aos observados antes da inoculação a partir do 23o DPI. Houve diminuição na liberação de H2O2 no segundo DPI, e no 16o DPI esses valores retornaram aos observados antes da inoculação. O declínio da atividade fagocítica dos MA, na fase aguda da infecção, indicou que essas células atuam de forma importante nos mecanismos de defesa pulmonar contra esse agente.
Palavras-chave: eqüino, macrófago alveolar, herpesvírus eqüino, EHV-1, lavado broncoalveolar
Equine rhinopneumonitis, caused by equine herpesvirus-1 (EHV-1) and equine herpesvirus-4 (EHV-4) is one of the main respiratory diseases in horses, leading to economical losses in athletic animals, due to treatment costs and to impaired athletic performance (Allen, Bryans, 1986).
Infectious diseases, such as the ones caused by respiratory viruses, may decrease the function of pulmonary alveolar macrophages (PAM) (Jakab, 1982; Nikerson, Jakab, 1990). Pulmonary viral infections may predispose animals and humans to secondary bacterial infections (Jakab, 1982; Nikerson, Jakab, 1990). Evidence is accumulating that, in the absence of other immunosuppressor factors, the virus may be responsible for the decrease in PAM function, affecting phagocytosis and bacterial death (Jakab, 1982; Nikerson, Jakab, 1990).
One of the main defense mechanisms of the lower respiratory tract against microorganisms and other inhaled particles is related to PAM phagocytic ability (Laegreid et al., 1988; Wong et al., 1990). PAM have an important role in EHV-1 infection in horses, influencing both the evolution of the disease and the recovery phase. The elucidation of innate immune responses has been carried out mainly in mice and humans, and there is currently little specific information about responses to viral infections in horses (Slater, Hannant, 2000). Apart from their important role in the inflammatory process and as phagocytic scavengers of infectious agents, macrophages stimulate the antiviral activity of NK and T cells through the production of IL-12 (Karupiah et al., 1996). The synthesis of cytokines by PAM may also be altered by EHV-1 infection. Cytokines are produced by mononuclear cells and virus-infected cells. They are key molecules in the innate immune response and influence the development of the adaptive immune response (Slater, Hannant, 2000).
PAM activity in healthy horses has been previously studied by spreading (chemotaxis), zymosan particle uptake (phagocytosis) and hydrogen peroxide (H2O2) production (respiratory burst) measurement (Mori, 2000; Mori et al., 2001). The objective of the present trial was to determine the effects of experimental EHV-1 infection on the alveolar leukocytes recovered by lavage from equine lungs in PAM functions in vitro.
MATERIALS AND METHODS
Five adult horses (four males and one female), of unknown breed, from 5 to 16 years old, weighting 332-415kg were used. Animals were kept in individual 3.5´3.5 meter enclosures, in the horse experimental sector of Departamento de Clínica Médica da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo. Animals were fed a total of 4kg of pelleted concentrates and 4kg of dust-free hay for each animal, twice a day; water was offered ad libitum. Bed consisted of pinus chips and was changed daily. These management conditions for the animals are in accordance with the recommendations described in Canadian (1993). For four months prior to challenge, horses were maintained stabled and showed no seroconversion in EHV-1 neutralizing antibody titers before the experimental inoculation (Table 1). There were not previous histories of experimental infections or vaccination. At the beginning of the study, none of the horses had overt signs of respiratory or other diseases. Rectal temperature and clinical signs were recorded daily during the acute stage of EHV-1 infection for 12 days.
EHV-1 used was derived from the standard A4/72 sample (Instituto Biológico de São Paulo, São Paulo SP, Brazil) originally isolated from an equine fetus aborted and cultured by 15 passages in VERO cells. Viral inoculum used in the challenge was 1ml (106.6 TCID50/ml) of EHV-1 by direct intranasal instillation in the rostral turbinates.
In the EHV-1 serological diagnosis, viral neutralization microtechnique described by Kotait et al. (1989) in VERO cells was used for the detection of virus specific serum antibodies, using the A4/72 sample of EHV-1 (100 TCID50/25µl) as antigen.
Nasal swabs were collected on the second, third and fifth days post-inoculation (DPI) with EHV-1. These samples were kept in Eagle medium with 10% of FCS (fetal calf serum) and after that they were inoculated in a culture of VERO cells (Saxegaard, 1966).
A bronchoalveolar lavage (BAL) silicon catheter (Bivona) measuring 10mm external diameter, 2.5mm internal diameter and 300cm long, was introduced from the nostril to the small bronchi, with the animal in a standing position and head and neck uplifted. Animals were sedated IV using a 0.44mg/kg xylazine associated with 0.033mg/kg butorphanol. A sterile PBS solution containing no Ca2+ or Mg2+ associated with 5UI/ml sodium heparin was introduced in the catheter in 5´60ml aliquots (total volume 300ml), and immediately aspirated. The volume recovered of lavage fluid was 76.0± 12.0%. BAL was performed in 7-day intervals. Samples were collected according to the description by Kydd et al. (1996). Before the challenge, four successive lavages were performed (28, 21, 14 and 7 days) in order to establish basal values for the variation in the number of cells and PAM activity. After experimental infection with EHV-1, BAL was performed on days +2, +9, +16, +23 and +30.
Aliquots of 200µl of cell suspension samples were cytocentrifugated (28g for six minutes), for the production of glass slides. Differential cell counts were determined by examination of 300 consecutive leukocytes in a preparation with Rosenfeld (Fernandes et al., 2000).
All BAL samples were centrifuged at 450g for 20 minutes, at 4° C. Supernatant was discarded and cells were resuspended in 1ml of RPMI 1640 associated to 10% of FCS. Cell viability and concentration was evaluated by trypan blue. After this procedure, cell concentration was adjusted for 2´106 cells/ml, number required for each of the three macrophage activity measurement tests.
Spreading assay, zymosan particle uptake (phagocytosis assay) and hydrogen peroxide (H2O2) release rate were conducted according to the method described by Mori (2000) and Mori et al. (2001).
Data analysis was performed using the statistical software GraphPad InStat® version 3.01, 32 bit for Windows 95/NT. Results of each trial were analyzed by comparison among means and representative standard deviations in the different post-inoculation days. The statistical test used in the evaluation of BAL cytology, of spreading, phagocytosis and H2O2 release data was the variance analysis (ANOVA). Student's t test was used in the comparison of the paired samples in the H2O2 release test, with and without PMA (P<0.05).
On the first two days after the inoculation with EHV-1, animals presented eye discharge and congestion of eyelid conjunctiva. Little serous, bilateral, nasal discharge was also observed. After seven days of the inoculation, this secretion became mucous. Account respiratory rates remained inside the normal range described for the species. Thorax auscultation showed normal lung sounds. No periods of increase in body temperature were observed during the whole trial.
A significant (P<0.05) increase in serum EHV-1 neutralizing antibodies was observed in the inoculated horses by 7DPI, when compared to day 0 (zero). Virus-neutralizing antibody titers remained significantly elevated throughout the sampling period (Table 1).
All animals were positive in nasal secretion viral isolation inoculated in VERO cells, on the second, third and fifth DPI.
In BAL cytology, macrophages were the predominant cell type observed. After them, lymphocytes were the largest group. Neutrophils, eosinophils and mast cells were observed in lower numbers.
There was a significant (P<0.05) difference in relation to differential cell counts on the 16th DPI with EHV-1. On that day, there were a decrease in the number of macrophages and an increase in the number of alveolar lymphocytes when compared to days 14 and 7 (Table 2). There was a significant (P<0.05) increase in total cell counts on the 23rd DPI, when compared to day 7.
There was a significant (P<0.05) increase in the spreading rate on day 23 post-inoculation, when compared to days 28, 21, 14 and 7. Spreading rate on the 30th DPI was significantly (P<0.05) greater when compared to days 28 and 21 (Table 3).
The results demonstrated a significant (P<0.05) decrease in the phagocytosis rate on days 2, 9 and 16 after EHV-1 inoculation when compared to day 7. Significant (P<0.05) differences also occurred on day 9 post-inoculation when compared to day 30 (Table 3).
There were no significant differences in the release of hydrogen peroxide (H2O2) among days post-inoculation and pre-inoculation, both in cells with or without in vitro stimulation with PMA (Table 4). In the comparison of paired samples with or without PMA, no statistical difference was observed in Student's t test (P<0.05).
The evidence of infection by EHV-1 in inoculated animals was based on the results obtained in the serological diagnosis and viral isolation from the secretion in nasal cavity of each horse. It is known that EHV-1 replicates in the respiratory mucosa during the initial phase of the disease and causes a large quantity of the infectious agent to be eliminated in nasal secretion (Mumford, 1994), and that the degree of tissue lesion depends on the quantity of antibodies available in the site of viral replication (Allen, Bryans, 1986).
The serum-neutralizing antibody persists for more than a year following natural or experimental infection, particularly in older horses, and most animals of two years and older have antibodies to EHV-1 (Bryans, Allen, 1989). The serum-neutralizing antibodies tests are useful for diagnosis of rhinopneumonitis only when acute and convalescent samples are available for testing. An increase of 2 log10 dilution in virus neutralizing titer indicates current infection with EHV-1 (Bryans, Allen, 1989).
After the inoculation with EHV-1, seroconversion of antibody titers was observed in the recovery phase (after day 14). This result is similar to that described by Stokes et al. (1991) for adult animals submitted to anamnestic immune response. In older horses that have had previous contact with the virus, viremia lasts for 5-7 days and the respiratory tract tissue in not so extensively affected that the disease may be recognized (Allen, Bryans, 1986).
According to previous studies (Mori, 2000; Fernandes et al., 2000; Mori et al., 2001), little variation in cell types was observed in BAL from healthy horses. Dynamic leukocyte changes in the bronchoalveolar compartment, such as neutrofilia, lymphopenia and a decrease in PAM were observed by Kydd et al. (1996), on the second day post inoculation with EHV-1. These changes were not observed in the present trial. There was a significant increase in the number of lymphocytes and decrease in the number of PAM only on day 16 post-inoculation (Table 2). The increase in the number of lymphocytes in BAL is probably due to cellular immune response of T-lymphocytes against viral infection (Freeman et al., 1993). Kydd et al. (1996) observed an increase in CD8+ CTL counts in horse BAL on the 21st DPI with EHV-1. It is possible that these lymphocytes are stimulated by viral infection; they identify and eliminate cells infected by the virus and, therefore, promote cell immunity against EHV-1 (Smith et al., 1998; O'Neill et al., 1999).
Repeated lavages could potentially influence pulmonary cell populations. It is well known that the sampling procedure in lungs induces neutrophilia lasting three-four days (Traub-Dargatz et al., 1988; McGorum, Dixon, 1994). Bielefeldt-Ohmann and Babiuk (1986b) also observed that successive lavages in 48-hour intervals led to a significant influx of lymphocytes. In the present trial, BAL collections were performed every seven days in all horses in order to eliminate the iatrogenic effects of the lavages on alveolar cell composition and possibly on PAM function, according to what was described by several authors (Bielefeldt-Ohmann, Babiuk, 1986b; Wong et al., 1990; Clark et al., 1995).
The percentages of neutrophils in BAL fluids obtained from control horses in this study were highly comparable with some of the previously published data (McGorum et al., 1993; McGorum, Dixon, 1994), but lower than the value reported by Tremblay et al. (1993). However, similar values were observed by Holcombe et al. (2001), in a study with stabled horses. The authors suggest that influx of neutrophils into airways occurs in healthy animals as a defense reaction to dust exposure in stabling and that stabling can contribute to airway inflammation. Thus, in order to decrease the possible environmental effects on alveolar cell population, some measures were adopted, such as, stabling animals for four months before the viral challenge, daily bed change and feeding with pelleted concentrated and dust-free hay.
The spreading rate (chemotaxis) of PAM in horses inoculated with EHV-1 had a significant increase on day 23 post-inoculation, which indicates a greater activation of these phagocytes in relation to the days before inoculation (Table 3). This PAM activation may probably occur due to the release of cytokines, such as gamma interferon, by CD4+ T cells (Smith et al., 1998). Macrophages then became activated and resistant to reinfection during the recovery phase. Activated macrophages, on their turn, have greater phagocytic activity - mainly in relation to oxidative metabolism in the elimination of microorganisms, and have an important role in the lung defense mechanism (Laegreid et al., 1988; Wong et al., 1990).
The phagocytosis rate of PAM decreased on days 2, 9 and 16 after virus inoculation, what presents statistical difference in relation to day 7. Reduction in PAM phagocytic ingestion on the first days post-inoculation was also observed in studies using several species in vivo and in vitro (Forman, Babiuk, 1982; Brown, Ananaba, 1988; Brown, Shin, 1990). After this period, PAM activity was recovered up to phagocytosis rates similar to those observed before the inoculation with EHV-1.
The reduction of H2O2, as detected by the respiratory burst assay on the second and ninth DPI, was not statistically significant. Reduction in PAM antimicrobial activity in the post-inoculation period has been reported (Bielefeldt-Ohmann, Babiuk, 1986a; Nickerson, Jakab, 1990; Chiou et al., 2000) both in in vitro and in vivo studies using different species. After this period of decrease in antimicrobial activity, there was a recovery in PAM function, whose activity was similar to that observed before inoculation. These results showed a decline in PAM activity shortly after infection. Similar results were observed in a study (Bridges, Edington, 1986) of blood monocytes from adult horses infected with EHV-1, in which a decrease in phagocytic ingestion from second and 10th DPI and in antimicrobial activity on the ninth DPI were reported, possibly due to viral replication in mononuclear cells. Experimental trials have demonstrated that during the virus infection, the antibacterial defenses of the lungs are suppressed through dysfunctions of the PAM phagocytic system (Nickerson, Jakab, 1990; Chiou et al., 2000).
EHV-1 infection leads to a decrease in PAM phagocytic and microbiocidal (respiratory burst) activity during the acute phase of the disease, probably due to viral replication inside the macrophages. After three weeks of viral infection, there is an increase in PAM chemotatic activity, what suggests that these cells may have an important role in the recovery period, preventing the occurrence of secondary infections, such as pneumonia. The mechanisms by which cell immune response in horses infected by EHV-1 occur require further studies, and the understanding of these mechanisms is essential for the development of efficient vaccines.
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Recebido para publicação em 24 de outubro de 2002
Recebido para publicação, após modificações, em 3 de abril de 2003