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Pulmonary intravascular macrophages in the pathogenesis of bovine pulmonary lesions caused by Actinomyces pyogenes
J. Comp. Path. 1995 Vol. 112, 197 206
Pulmonary Intravascular Macrophages in the Pathogenesis of Bovine Pulmonary Lesions Caused by Actinomyces pyogenes P. S. Leifsson, A. Basse, H. E. J e n s e n , B. Bloch* and B. Aalbmk* Department of Pharmacology and Pathobiology and *Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University Copenhagen, BMowsvej 13, DK-1870 Frederiksberg C, Denmark
Summary Rabbit antisera raised against somatic antigens from two strains of Actinomyces pyogenes reacted specifically in a peroxidase anti-peroxidase technique which was developed for the location of the bacteria in formalin-fixed tissues. The technique was applied on experimental murine and spontaneous bovine lesions caused by A. pyogenes. By electron microscopy and immunohistochemistry it was demonstrated that pulmonary intravascular macrophages play a role in the uptake of A. pyogenes from the blood, and in the production of pyaemic pulmonary lesions.
Introduction Pulmonary intravascular macrophages (PIMs) are members of the mononuclear phagocyte system and are abundant in the pulmonary capillaries of ruminants, swine, cats and horses, in which they adhere to the endothelial cells (Schneeberger-Keeley and Burger, 1970; Rybicka et al., 1974; Warner and Brain, 1984a,b; Winkler and Cheville, 1984; Warner et al., 1986; Atwal et al., 1992). PIMs have also been identified in rabbits, baboons, rats, dogs and man, but to a lesser extent, and their importance as phagocytes in these species is apparently overshadowed by the macrophages of the liver (Kupffer cells) and spleen (Francica et al., 1988; Schultz et al., 1988; Dehring and Wismar, 1989; Niehaus, 1989; Warner et al., 1989). In recent years the immunological properties of PIMs and their role in pulmonary diseases have been partly elucidated, especially in relation to their clearance of circulating particles and soluble agents (Brain et al., 1988; Bertram et al., 1989). The uptake of Gram-negative bacteria and endotoxin by PIMs "triggers" the release of vasoactive and inflammatory mediators in vitro; this phenomenon results in acute pulmonary injury in animals with Gram-negative sepsis (Dehring et al., 1983; Chitko-McKown et al., 1992). The role of PIMs in the pathogenesis of Gram-positive infections has not been clarified. However, intravenously injected suspensions of Staphylococcus aureus cause significant pulmonary hypertension in pigs and the bacteria can be found within phagosomes of intravascular macrophages, but only minor 0021 9975/95/020197 + 10 $08.00/0
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morphological changes in the lungs have been observed (Dehring et al., 1983). In spite of the importance ofActinomyces pyogenes as a pathogen of ruminants and pigs, studies of the pathogenesis of infections produced by this organism have been largely neglected (Songer and Prescott, 1993). Thrombosis of the pulmonary vessels, especially arteries, is common in cattle infected with A. pyogenes and often leads to infarction and abscess formation (unpublished observations). In the present paper we report on a case of pyaemic infection in a cow caused by A. pyogenes, with emphasis on the immunohistochemical and electron microscopical findings in the lungs, especially the uptake of the bacteria by PIMs. Materials and Methods
Antigen Two strains (nos 7770 and 7773) ofA. pyogenes isolated from cases of bovine endocarditis were used for the production of antigen. Bacteria were harvested from Tryptose Blood Agar Base (Difco Laboratories, USA), containing ox blood 5%, after anaerobic incubation (H2 90%, CO2 10%) at 37~ for 48 h, and suspended in 0"9% saline. After sedimentation at 5~ for 18 h and removal of the supernates, the bacterial suspensions were homogenized by an X-press (AB Biox, Sweden) with a pre-cooled (-30~ 25-ml cylinder operated with a maximal force of 200 MPa (]ensen et al., 1993). The products were centrifuged at 20 000g and 5~ for 1 h, and the supernates were recovered. After sterile filtration (Minisart NML, Sartorius, Germany) these preparations (somatic antigen) were stored in aliquots at --80~ The protein content of somatic antigens nos 7770 and 7773 was 6-9 mg/ml and 3'0 mg/ml, respectively, as determined by the modified Lowry method (Markwell et al., 1978).
Antisera Two New Zealand white rabbits (3 kg) were immunized, one with four monthly subcutaneous injections of 0'5 ml of antigen 7770 preparation emulsified with 0'5 ml of Freund's complete adjuvant, and the other with similar injections of antigen 7773. During the immunization period the immune response was evaluated by crossed immunoelectrophoresis. Eight days after the fourth injection, the animals were bled and the hyperimmune sera were recovered and stored in aliquots at -25~
Crossed Immunoelectrophoresis Crossed immunoelectrophoresis was done as described by Weeke (1973) and Jensen et al. (1990). Antisera were used in 10% (v/v) dilutions and the antigens were applied in volumes of 5 l.tl (3-0 mg protein/ml). The antisera were tested with their homologous and heterologous antigenic preparations, and after electrophoresis the number of immuno-precipitates was recorded.
Murine Tissues Tissues from two mice experimentally infected with A. pyogenes (strain 7770 or 7773) were used for the evaluation of immunohistochemical staining. The mice were inoculated intravenously with a suspension of bacteria (c. 10t~ and killed 5 min later. Tissues were removed and processed for histopathological examination. Only tissues in which abundant numbers of bacteria were seen by Gram-staining were used
Bovine PIMs and A. pyogenes
199
for the evaluation of the plateau and staining end-points in the peroxidase antiperoxidase (PAP) technique (see below). Tissues from two uninfected mice were used as controls.
Bovine 7-issues Various tissues from a cow with a systemic infection, found to be due to A. pyogenes, were examined. The pulmonary lesions consisted of thrombosis of the main branches of the pulmonary artery, multiple haemorrhagic infarcts, and abscesses. The cow had thrombotic endocarditis on all valves, multiple infarcts of the kidneys, infarction of the spleen and decubital lesions on both hocks. Tissue samples from organs with macroscopical lesions were collected and processed for histopathological examination, but only those from the heart valves and lungs are dealt with here. As a control, lung tissue from a cow with Johne's disease was examined.
Bacteriology Samples were collected aseptically from the spleen, kidneys, heart valves and lungs of the pyaemic cow and the lungs of the cow with Johne's disease. The samples were cultured on Tryptose Blood Agar Base (Difco Laboratories, USA), containing ox blood 5%, and incubated anaerobically (H2 90%, CO2 10%) at 37~ for 48 h, and also aerobically. The organism isolated from the pyaemic cow consisted of non-motile, catalase-negative, Gram-positive polymorphous rods forming small, slowly growing, smooth colonies with fl-haemolysis, stimulated by carbon dioxide, fermenting glucose and hydrolysing protein (Loeffler serum slopes); it was identified as A. pyogenes by the methods of Barrow and Feltham (1993).
Histopathology Tissues were fixed in 10% buffered formalin for 5 to 7 days, dehydrated, embedded in paraffin wax, cut in sections 4 to 5 gm thick, and mounted on StarFrost| adhesive slides (AxelJohnson Lab System, Denmark). Sections from infected and control mice were stained with haematoxylin and eosin (HE) and Gram stain. All bovine tissues were stained with HE, Mallory's phosphotungstic acid-haematoxylin (PTAH) and Gram stain.
Immunohistochemical Examination of Murine 7~ssues The principles of the peroxidase anti-peroxidase (PAP) technique were described by Jensen et al. (1993). In its present form serial two-fold dilutions of each of the two hyperimmune sera were tested against the homologous and the heterologous bacterial strain, and the plateau and staining end-points were determined (Jensen and Schonheyder, 1989). As controls the hyperimmune sera were tested on uninfected murine tissues, and were replaced by a preimmune and a hyperimmune serum of unrelated specificity (rabbit anti-Candida krusei serum). In other experiments the hyperimmune sera were absorbed with an equal volume of homologous antigen preparation or replaced by normal swine serum 5% (v/v). As the 7770 antiserum revealed the higher plateau end-point, it alone was used for the staining of bovine tissues (below).
Immunohistochemical Examination of Bovine 77ssues As the initial dilutions of the 7770 antiserum used for the PAP-labelling of bovine tissues showed some non-specific staining at the plateau end-point (1 in 256), it was diluted further to 1 in 1024. This proved suitable for the screening of tissues.
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P . S . L e i f s s o n e t al.
Table 1 Examination of two A. p y o g e n e s antigen preparations and their corresponding antisera by the crossed i m m u n o e l e c t r o p h o r e s i s and peroxidase/anti-peroxidase techniques Results *~, of tests with Antigen 7770 Antiserum
Antigen 7773
XIE*
PEI ~
SEPt+
XIE*
PEI~
SEPt+
37 39
1 in 256 1 in 32
1 in 4096 1 in 512
39 37
1 in 256 1 in 64
1 in 8192 1 in 4096
7770 7773
* N u m b e r of immunoprecipitates in crossed immunoelectrophoresis. "~Plateau end-point (dilution). ++Staining end-point (dilution).
Electwn Microscopy (EM) Samples from the bovine lungs were fixed in glutaraldehyde 2-5% in 0" 13 M phosphate buffer and postfixed in osmium tetroxide 1% in the same buffer. After dehydration in acetone the samples were embedded in Vestopal-W (Serva, Germany). Sections were stained with toluidine blue, and thin sections for electron microscopy were contrasted with uranyl acetate and lead citrate.
Results
Crossed Immunoelectrophoresis The two antisera raised against A. pyogenes cross-reacted strongly, and, surprisingly, the number of immunoprecipitates in the heterologous tests proved higher than those in homologous tests (Table 1). Quantitative and qualitative differences in the reactions of the antisera were not observed.
Murine 77ssues Gram-positive bacteria were found mainly within the lumen of the pulmonary vessels when H E and Gram-stained sections were evaluated. In the PAPstained sections the bacteria were stained intensely with both antisera, whereas no staining was observed with the control sera or in normal murine tissues. The plateau and staining end-points of the antisera are given in Table 1. The plateau end-points of antiserum 7770 were four- to eight-fold higher than those of antiserum 7773 in both homologous and heterologous reactions. The antisera had identical staining end-points when tested with homologous bacterial strains, but when tested with the heterologous strains antiserum 7770 had a staining end-point 16-fold higher than that of antiserum 7773. At high concentrations (<1 in 16) both antisera showed some non-specific staining, but at further dilutions (> 1 in 32) this gradually declined and specific staining was seen at dilutions at or higher than their plateau end-points.
B o v i n e PIMs and A. pyogenes
Fig. 1.
201
Lung tissue from a cow with A. pyogenes infection. Pulmonary intravascular macrophages (PIMs) containing A. pyogenes bacteria (arrows) are situated in the periphery of two capillaries (C). Alveoli (A). PAR Bar = 20 gin.
Bovine 7~ssues
In the lung sections the interalveolar and interlobular septa were thickened due to oedema and infiltration of mononuclear cells. Some lobules were necrotic and several abscesses were found. These consisted of accumulations of degenerate leucocytes, often surrounded by zones of macrophages and fibroblasts. In the abscesses Gram-positive bacteria were seen intracellularly and extracellularly. However, in a higher number of cells Gram-negative material, probably bacteria, was found. Thrombosis of the lymph vessels in the interlobular septa was observed in several sections. M a n y alveoli contained a proteinaceous fluid and some neutrophils. Several thrombi were seen within the pulmonary capillaries by PTAH staining. In bovine tissues non-specific staining by the PAP-technique was more persistent than in the murine tissues, but when the serum was further diluted (up to four-fold higher than the plateau end-point) this disadvantage was removed. The working dilution of 1 in 1024 provided an intense staining of the bacteria with only a faint non-specific staining of the periphery of sections. In the lung sections m a n y cells in the alveolar septa were stained (Fig. 1). Generally the precise location of the cells could not be determined, but occasionally it was evident that they were intravascular and adherent to the
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P.S. Leifsson e t al.
capillary endothelium. The cells were irregularly shaped, 15 to 20 gm in diameter and their nuclei varied in size and shape. The extent of the staining was different from cell to cell. In most cases, immunoreactivity was limited to one.or more spherical structures, 2 to 4 gm in diameter, containing granular material within the cytoplasm. In other cells similar granular material was found dispersed in the cytoplasm or the cells were distended with positively staining material. The number of immunoreactive cells in different parts of the lungs was constant. In the HE-stained sections of the heart valves numerous bacterial colonies were observed in the thrombi. O n the valves, granulation tissue heavily infiltrated with large macrophages was separated from the thrombi by zones of degenerate leucocytes. In the thrombi, bacteria in the centre of colonies tended to be Gram-positive whereas those in the periphery were Gramnegative. The macrophages in the granulation tissue contained small amounts of Gram-negative material. The bacterial colonies within all parts of the valvular thrombus material stained intensely when the PAP-technique was applied. Moreover, the macrophages in the granulation tissue were distended with immunoreactive material.
Bacteriology A. pyogenes was isolated in pure culture from the lungs, heart valves, spleen and kidneys. No bacteria were isolated from the lung tissue of the cow with Johne's disease.
Electron Microscopy (EM) Numerous mononuclear cells containing intact bacteria as well as distorted cell walls in their phagolysosomes were found in the pulmonary capillaries (Figs 2 and 3). The bacteria-containing cells often had a lobulated surface on which a fuzzy layer of stainable material adhered to a depth of approximately 17 nm from the cell membrane. Invaginations and infoldings from the coated surface formed structures described as micropinocytosis vermiformis (Fig. 2). Other parts of the cell surface were in intimate contact with the endothelium (Fig. 3). In m a n y cases the serum proteins in the capillaries in which the bacteria-containing cells were observed appeared highly condensed. Discussion
The two strains of A. pyogenes cross-reacted strongly in crossed immunoelectrophoresis, as might be expected from the observation that A. pyogenes is a serologically homogeneous species (Ryff and Browne, 1954; Sorensen, 1974). The development and application of a PAP-technique for the demonstration of A. pyogenes in tissues was specific on murine material at a primary antiserum dilution of 1 in 256 (plateau end-point). However, on bovine tissues some non-specific staining was observed at this dilution, but by
B o v i n e P I M s a n d A. p y o g e n e s
Fig. 2.
203
Part of a PIM lying between erythrocytes (E) in a lung capillary from a cow with pyaemia caused by A. pyogenes. Two bacteria can be seen in a lysosome (arrow) in the PIM. Note the presence of micropinocytosis vermiformis (M). EM. Bar ~ 1 gm.
a further four-fold dilution the primary antiserum was also rendered suitable for use on bovine tissues. The PAP-positive contents of macrophages in chronic cardiac lesions were usually not stained by the Gram method, and on the heart valves only some of the extracellular bacteria appeared Gram-positive, the remainder being Gram-negative. The cells ofA. pyogenes, especially when ageing, tend to become Gram-variable or negative (Sorensen, 1974; Gu6rin-Faubl~e et al., 1992). From their morphology, the bacteria-containing cells were identified as PIMs (Rybicka et al., 1974; Warner et al., 1986). The light and electron microscopical findings demonstrated that PIMs played a part in the uptake of A. pyogenes from the blood. The uptake of bacteria and particles by PIMs is followed by the release of a variety ofvasoactive and inflammatory substances, including thromboxanes and interleukin-1 (IL-1) (Miyamoto et al., 1987; Chitko-McKown et al., 1992). The thromboxanes are important mediators of lung microvascular injury, causing increased vascular pressure, increased neutrophil adherence to the endothelium, and pulmonary oedema (Malik et al., 1985). IL-1, a potent cytokine, has a central role in the inflammatory response. Among the activities ascribed to IL-1 are neutrophil activation, increased endothelial procoagulant activity, and increased adherence of neutrophils to the endothelium (Dinarello, 1992). Therefore, it is reasonable to assume that the accompanying inflammatory lung lesions in the present case
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P.S. Leifsson et al.
Fig. 3. Part of a PIM in a lung capillary. The cell contains large phagolysosomesin which a number of more or less decayed bacteria and bacterial walls can be seen. At both sides of the figure the alveoli and the alveolar epithelium (A) are shown. The PIM is in intimate contact with the endothelium (E) at the right side. Nucleus (N). EM. Bar = 1gin.
were caused b y the release o f vasoactive a n d i n f l a m m a t o r y substances f r o m the activated PIMs. In conclusion, b y PAP staining a n d electron m i c r o s c o p y it was d e m o n s t r a t e d that A. pyogenes cells m a y be taken up b y bovine P I M s . As well as clearing A. pyogenes f r o m the blood, the activated P I M s also a p p e a r e d to m e d i a t e thrombosis, infarction a n d inflammation, resulting in abscessation. T h u s , P I M s w o u l d seem to have an essential role in the initial stages o f the embolic p n e u m o n i a that occurs frequently in cattle with A. pyogenes p y a e m i a .
Acknowledgments This study was supported by the Icelandic Association of Dairy Farmers.
References Atwal, O. S., Singh, B., Staempfli, H. and Minhas, K. (1992). Presence of pulmonary intra-vascular macrophages in the equine lung: some structuro-functional properties. Anatomical Record, 234, 530-540. Barrow, G. I. and Feltham, R. K. A. (1993). Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd Edit., Cambridge University Press, Cambridge.
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Bertram, T. A., Overby, L. H., Brody, A. R, and Eling, T. E. (1989). Comparison of arachidonic acid metabolism by pulmonary intravascnlar and alveolar macrophages exposed to particulate and soluble stimuli. Laboratory Investigation, 61, 457-466. Brain, J. D., Warner, A. E., Molina, R. M. and DeCamp, M. M. (1988). Pulmonary intravascular macrophages are an important part of the mononuclear phagocyte system in ruminants and cats. American Review of Respirato~ Disease, 137, 147 (Abstract). Chitko-McKown, C. G., Reddy, D. N., Chapes, S. K., McKown, R. D. and Blecha, F. (1992). Immunological characterization of pulmonary intravascular macrophages. Regional Immunology, 4, 236 244. Dehring, D.J., Crocker, S. H., Wismar, B. L., Steinberg, S. M., Lowery, B. D. and Cloutier, C. T. (i983). Comparison of live bacteria infusions in a porcine model of acute respiratory failure. Journal of Surgical Research, 34, 151-158. Dehring, D.J. and Wismar, B. L. (1989). Intravascular macrophages in pulmonary capillaries of humans. American Review of Respiratory Disease, 139, 1027-1029. Dinarello, C. A. (1992). Role of interleukin-1 in infectious diseases. Immunological Reviews, 127, 119 146. Francica, R J., Bertram, T., Knapp, M. and Crapo, J. D. (1988). Pulmonary intravascular macrophages in normal and oxygen injured baboon lung tissue. Clinical Research, 36, 591A (Abstract). Gu~rin-Faubl6e, V., Karray, S., Tilly, B. and Richard, Y. (1992). Actinomycespyogenes: 6tude bacteriologique conventionelle et sur galeries Api de 103 souches isol6es chez les ruminants. Annales de Recherche Vdtdrinaire, 23, 151-160. Jensen, H. E., Aalbaek, B., Lind, R, Frandsen, R L., Krogh, H. V. and Stynen, D. (1993). Enzyme immunohistochemistry with mono- and polyclonal antibodies in the pathological diagnosis of systemic bovine mycoses. Acta PathologicaMicrobioIogica et Immunologica Scandinavica, 101,505 516. Jensen, H. E., Hau, J., Aalb~ek, B. and Schonheyder, H. (1990). Indirect immunofluorescence staining and crossed immunoelectrophoresis for differentiation of Candida albicans and Geotrichum candidum. Mycoses, 33, 519 526. Jensen, H. E. and Schonheyder, H. (1989). Immunofluorescence staining of hyphae in the histopathological diagnosis of mycoses in cattle. Journal of Medical and Veterinary Mycology, 27, 33-44. Malik, A. B., Perlman, M. B., Cooper, J. A., Noonan, T. and Bizios, R. (1985). Pulmonary microvascular effects of arachidonic acid metabolites and their role in lung vascular injury. Federation Proceedings, 44, 36-42. Markwell, M. A. K., Haas, S. M., Bieber, L. L. and Tolbert, N. E. (1978). A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Analytical Biochemistry, 87, 206-210. Miyamoto, K., Schultz, E., Mitchell, M. and Staub, N. C. (198'7). Systemic arterial thromboxane concentrations after liposome infusions in three species with pulmonary intracapillary macrophages. Federation Proceedings, 46, 1109 (Abstract). Niehaus, G. D. (1989). Role in systemic host defence. In: The Pulmona~ Intravascular Macrophage, N. C. Staub, Ed., Futura Publishing Company, Mount Kisco, New York, pp. 39-58. Rybicka, K., Daly, B. D. T., Migliore, J. j. and Norman, J. C. (1974). Intravascular macrophages in normal calf lung. An electron microscopic study. AmericanJournal of Anatomy, 139, 353-368. Ryff, J. F. and Browne, J. (1954). Corynebacteriumpyogenes--a cultural and serological study. AmericanJournal of Veterinary Research, 15, 617-621. Schneeberger-Keeley, E. E. and Burger, E.J. (1970). Intravascular macrophages in cat lungs after open chest ventilation. Laboratory Investigation, 22, 361-369. Schultz, E., Milligan, S., Yuen, C., Wang, Y., Enzan, K., Staub, N. C. and Goldstein, I. (1988). Comparison of pulmonary intravascular macrophages from goats and rabbits in primary culture. FASEBJournal, 2, A725 (Abstract).
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Songer, J. G. and Prescott, J. E (1993). Corynebacterium. In: Pathogenesis of Bacterial Infections in Animals, C. L. Gyles and C. O. Thoen, Eds, Iowa State University Press, Ames, pp. 57-69. Sorensen, G. H. (1974). Corynebacteriumpyogenes. A biochemical and serological study. Acta Veterinaria Scandinavica, 15, 544 554. Warner, A. E., Barry, B. E. and Brain, J. D. (1986). Pulmonary intravaseular macrophages in sheep. Morphology and function of a novel constituent of the mononuclear phagocyte system. Laboratory Investigation, 55, 276 288. Warner, A. E. and Brain, J. D. (1984a). The ruminant reticuloendothelial system includes phagocytic intravascular pulmonary macrophages. Journal of Leukocyte Biology, 36, 388 (Abstract). Warner, A. E. and Brain, J. D. (1984b). Intravascular pulmonary macrophages in ruminants participate in reticuloendothelial clearance of particles. Federation Proceedings, 43, 1001 (Abstract). Warner, A. E., DeCamp, M. M., Bellows, C. F. and Brain, J. D. (1989). Endotoxemia enhances lung uptake of circulating particles in species lacking pulmonary intravascular macrophages. American Review of Respiratory Disease, 139, A158 (Abstract). Weeke, B. (1973). Crossed immunoelectrophoresis. ScandinavianJournal of Immunology, 2, (Supplement 1), 47-56. Winlder, G. C. and Cheville, N. F. (1984). The neonatal porcine lung: ultrastructural morphology and postnatal development of the terminal airways and alveolar region. Anatomical Record, 211), 303 313.
Received, September 19th, 1994] Accepted, November 2nd, 1994 J
Pulmonary Intravascular Macrophages in the Pathogenesis of Bovine Pulmonary Lesions Caused by Actinomyces pyogenes P. S. Leifsson, A. Basse, H. E. J e n s e n , B. Bloch* and B. Aalbmk* Department of Pharmacology and Pathobiology and *Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University Copenhagen, BMowsvej 13, DK-1870 Frederiksberg C, Denmark
Summary Rabbit antisera raised against somatic antigens from two strains of Actinomyces pyogenes reacted specifically in a peroxidase anti-peroxidase technique which was developed for the location of the bacteria in formalin-fixed tissues. The technique was applied on experimental murine and spontaneous bovine lesions caused by A. pyogenes. By electron microscopy and immunohistochemistry it was demonstrated that pulmonary intravascular macrophages play a role in the uptake of A. pyogenes from the blood, and in the production of pyaemic pulmonary lesions.
Introduction Pulmonary intravascular macrophages (PIMs) are members of the mononuclear phagocyte system and are abundant in the pulmonary capillaries of ruminants, swine, cats and horses, in which they adhere to the endothelial cells (Schneeberger-Keeley and Burger, 1970; Rybicka et al., 1974; Warner and Brain, 1984a,b; Winkler and Cheville, 1984; Warner et al., 1986; Atwal et al., 1992). PIMs have also been identified in rabbits, baboons, rats, dogs and man, but to a lesser extent, and their importance as phagocytes in these species is apparently overshadowed by the macrophages of the liver (Kupffer cells) and spleen (Francica et al., 1988; Schultz et al., 1988; Dehring and Wismar, 1989; Niehaus, 1989; Warner et al., 1989). In recent years the immunological properties of PIMs and their role in pulmonary diseases have been partly elucidated, especially in relation to their clearance of circulating particles and soluble agents (Brain et al., 1988; Bertram et al., 1989). The uptake of Gram-negative bacteria and endotoxin by PIMs "triggers" the release of vasoactive and inflammatory mediators in vitro; this phenomenon results in acute pulmonary injury in animals with Gram-negative sepsis (Dehring et al., 1983; Chitko-McKown et al., 1992). The role of PIMs in the pathogenesis of Gram-positive infections has not been clarified. However, intravenously injected suspensions of Staphylococcus aureus cause significant pulmonary hypertension in pigs and the bacteria can be found within phagosomes of intravascular macrophages, but only minor 0021 9975/95/020197 + 10 $08.00/0
9 1995 Academic Press Limited
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P.S. Leifsson e t al.
morphological changes in the lungs have been observed (Dehring et al., 1983). In spite of the importance ofActinomyces pyogenes as a pathogen of ruminants and pigs, studies of the pathogenesis of infections produced by this organism have been largely neglected (Songer and Prescott, 1993). Thrombosis of the pulmonary vessels, especially arteries, is common in cattle infected with A. pyogenes and often leads to infarction and abscess formation (unpublished observations). In the present paper we report on a case of pyaemic infection in a cow caused by A. pyogenes, with emphasis on the immunohistochemical and electron microscopical findings in the lungs, especially the uptake of the bacteria by PIMs. Materials and Methods
Antigen Two strains (nos 7770 and 7773) ofA. pyogenes isolated from cases of bovine endocarditis were used for the production of antigen. Bacteria were harvested from Tryptose Blood Agar Base (Difco Laboratories, USA), containing ox blood 5%, after anaerobic incubation (H2 90%, CO2 10%) at 37~ for 48 h, and suspended in 0"9% saline. After sedimentation at 5~ for 18 h and removal of the supernates, the bacterial suspensions were homogenized by an X-press (AB Biox, Sweden) with a pre-cooled (-30~ 25-ml cylinder operated with a maximal force of 200 MPa (]ensen et al., 1993). The products were centrifuged at 20 000g and 5~ for 1 h, and the supernates were recovered. After sterile filtration (Minisart NML, Sartorius, Germany) these preparations (somatic antigen) were stored in aliquots at --80~ The protein content of somatic antigens nos 7770 and 7773 was 6-9 mg/ml and 3'0 mg/ml, respectively, as determined by the modified Lowry method (Markwell et al., 1978).
Antisera Two New Zealand white rabbits (3 kg) were immunized, one with four monthly subcutaneous injections of 0'5 ml of antigen 7770 preparation emulsified with 0'5 ml of Freund's complete adjuvant, and the other with similar injections of antigen 7773. During the immunization period the immune response was evaluated by crossed immunoelectrophoresis. Eight days after the fourth injection, the animals were bled and the hyperimmune sera were recovered and stored in aliquots at -25~
Crossed Immunoelectrophoresis Crossed immunoelectrophoresis was done as described by Weeke (1973) and Jensen et al. (1990). Antisera were used in 10% (v/v) dilutions and the antigens were applied in volumes of 5 l.tl (3-0 mg protein/ml). The antisera were tested with their homologous and heterologous antigenic preparations, and after electrophoresis the number of immuno-precipitates was recorded.
Murine Tissues Tissues from two mice experimentally infected with A. pyogenes (strain 7770 or 7773) were used for the evaluation of immunohistochemical staining. The mice were inoculated intravenously with a suspension of bacteria (c. 10t~ and killed 5 min later. Tissues were removed and processed for histopathological examination. Only tissues in which abundant numbers of bacteria were seen by Gram-staining were used
Bovine PIMs and A. pyogenes
199
for the evaluation of the plateau and staining end-points in the peroxidase antiperoxidase (PAP) technique (see below). Tissues from two uninfected mice were used as controls.
Bovine 7-issues Various tissues from a cow with a systemic infection, found to be due to A. pyogenes, were examined. The pulmonary lesions consisted of thrombosis of the main branches of the pulmonary artery, multiple haemorrhagic infarcts, and abscesses. The cow had thrombotic endocarditis on all valves, multiple infarcts of the kidneys, infarction of the spleen and decubital lesions on both hocks. Tissue samples from organs with macroscopical lesions were collected and processed for histopathological examination, but only those from the heart valves and lungs are dealt with here. As a control, lung tissue from a cow with Johne's disease was examined.
Bacteriology Samples were collected aseptically from the spleen, kidneys, heart valves and lungs of the pyaemic cow and the lungs of the cow with Johne's disease. The samples were cultured on Tryptose Blood Agar Base (Difco Laboratories, USA), containing ox blood 5%, and incubated anaerobically (H2 90%, CO2 10%) at 37~ for 48 h, and also aerobically. The organism isolated from the pyaemic cow consisted of non-motile, catalase-negative, Gram-positive polymorphous rods forming small, slowly growing, smooth colonies with fl-haemolysis, stimulated by carbon dioxide, fermenting glucose and hydrolysing protein (Loeffler serum slopes); it was identified as A. pyogenes by the methods of Barrow and Feltham (1993).
Histopathology Tissues were fixed in 10% buffered formalin for 5 to 7 days, dehydrated, embedded in paraffin wax, cut in sections 4 to 5 gm thick, and mounted on StarFrost| adhesive slides (AxelJohnson Lab System, Denmark). Sections from infected and control mice were stained with haematoxylin and eosin (HE) and Gram stain. All bovine tissues were stained with HE, Mallory's phosphotungstic acid-haematoxylin (PTAH) and Gram stain.
Immunohistochemical Examination of Murine 7~ssues The principles of the peroxidase anti-peroxidase (PAP) technique were described by Jensen et al. (1993). In its present form serial two-fold dilutions of each of the two hyperimmune sera were tested against the homologous and the heterologous bacterial strain, and the plateau and staining end-points were determined (Jensen and Schonheyder, 1989). As controls the hyperimmune sera were tested on uninfected murine tissues, and were replaced by a preimmune and a hyperimmune serum of unrelated specificity (rabbit anti-Candida krusei serum). In other experiments the hyperimmune sera were absorbed with an equal volume of homologous antigen preparation or replaced by normal swine serum 5% (v/v). As the 7770 antiserum revealed the higher plateau end-point, it alone was used for the staining of bovine tissues (below).
Immunohistochemical Examination of Bovine 77ssues As the initial dilutions of the 7770 antiserum used for the PAP-labelling of bovine tissues showed some non-specific staining at the plateau end-point (1 in 256), it was diluted further to 1 in 1024. This proved suitable for the screening of tissues.
200
P . S . L e i f s s o n e t al.
Table 1 Examination of two A. p y o g e n e s antigen preparations and their corresponding antisera by the crossed i m m u n o e l e c t r o p h o r e s i s and peroxidase/anti-peroxidase techniques Results *~, of tests with Antigen 7770 Antiserum
Antigen 7773
XIE*
PEI ~
SEPt+
XIE*
PEI~
SEPt+
37 39
1 in 256 1 in 32
1 in 4096 1 in 512
39 37
1 in 256 1 in 64
1 in 8192 1 in 4096
7770 7773
* N u m b e r of immunoprecipitates in crossed immunoelectrophoresis. "~Plateau end-point (dilution). ++Staining end-point (dilution).
Electwn Microscopy (EM) Samples from the bovine lungs were fixed in glutaraldehyde 2-5% in 0" 13 M phosphate buffer and postfixed in osmium tetroxide 1% in the same buffer. After dehydration in acetone the samples were embedded in Vestopal-W (Serva, Germany). Sections were stained with toluidine blue, and thin sections for electron microscopy were contrasted with uranyl acetate and lead citrate.
Results
Crossed Immunoelectrophoresis The two antisera raised against A. pyogenes cross-reacted strongly, and, surprisingly, the number of immunoprecipitates in the heterologous tests proved higher than those in homologous tests (Table 1). Quantitative and qualitative differences in the reactions of the antisera were not observed.
Murine 77ssues Gram-positive bacteria were found mainly within the lumen of the pulmonary vessels when H E and Gram-stained sections were evaluated. In the PAPstained sections the bacteria were stained intensely with both antisera, whereas no staining was observed with the control sera or in normal murine tissues. The plateau and staining end-points of the antisera are given in Table 1. The plateau end-points of antiserum 7770 were four- to eight-fold higher than those of antiserum 7773 in both homologous and heterologous reactions. The antisera had identical staining end-points when tested with homologous bacterial strains, but when tested with the heterologous strains antiserum 7770 had a staining end-point 16-fold higher than that of antiserum 7773. At high concentrations (<1 in 16) both antisera showed some non-specific staining, but at further dilutions (> 1 in 32) this gradually declined and specific staining was seen at dilutions at or higher than their plateau end-points.
B o v i n e PIMs and A. pyogenes
Fig. 1.
201
Lung tissue from a cow with A. pyogenes infection. Pulmonary intravascular macrophages (PIMs) containing A. pyogenes bacteria (arrows) are situated in the periphery of two capillaries (C). Alveoli (A). PAR Bar = 20 gin.
Bovine 7~ssues
In the lung sections the interalveolar and interlobular septa were thickened due to oedema and infiltration of mononuclear cells. Some lobules were necrotic and several abscesses were found. These consisted of accumulations of degenerate leucocytes, often surrounded by zones of macrophages and fibroblasts. In the abscesses Gram-positive bacteria were seen intracellularly and extracellularly. However, in a higher number of cells Gram-negative material, probably bacteria, was found. Thrombosis of the lymph vessels in the interlobular septa was observed in several sections. M a n y alveoli contained a proteinaceous fluid and some neutrophils. Several thrombi were seen within the pulmonary capillaries by PTAH staining. In bovine tissues non-specific staining by the PAP-technique was more persistent than in the murine tissues, but when the serum was further diluted (up to four-fold higher than the plateau end-point) this disadvantage was removed. The working dilution of 1 in 1024 provided an intense staining of the bacteria with only a faint non-specific staining of the periphery of sections. In the lung sections m a n y cells in the alveolar septa were stained (Fig. 1). Generally the precise location of the cells could not be determined, but occasionally it was evident that they were intravascular and adherent to the
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capillary endothelium. The cells were irregularly shaped, 15 to 20 gm in diameter and their nuclei varied in size and shape. The extent of the staining was different from cell to cell. In most cases, immunoreactivity was limited to one.or more spherical structures, 2 to 4 gm in diameter, containing granular material within the cytoplasm. In other cells similar granular material was found dispersed in the cytoplasm or the cells were distended with positively staining material. The number of immunoreactive cells in different parts of the lungs was constant. In the HE-stained sections of the heart valves numerous bacterial colonies were observed in the thrombi. O n the valves, granulation tissue heavily infiltrated with large macrophages was separated from the thrombi by zones of degenerate leucocytes. In the thrombi, bacteria in the centre of colonies tended to be Gram-positive whereas those in the periphery were Gramnegative. The macrophages in the granulation tissue contained small amounts of Gram-negative material. The bacterial colonies within all parts of the valvular thrombus material stained intensely when the PAP-technique was applied. Moreover, the macrophages in the granulation tissue were distended with immunoreactive material.
Bacteriology A. pyogenes was isolated in pure culture from the lungs, heart valves, spleen and kidneys. No bacteria were isolated from the lung tissue of the cow with Johne's disease.
Electron Microscopy (EM) Numerous mononuclear cells containing intact bacteria as well as distorted cell walls in their phagolysosomes were found in the pulmonary capillaries (Figs 2 and 3). The bacteria-containing cells often had a lobulated surface on which a fuzzy layer of stainable material adhered to a depth of approximately 17 nm from the cell membrane. Invaginations and infoldings from the coated surface formed structures described as micropinocytosis vermiformis (Fig. 2). Other parts of the cell surface were in intimate contact with the endothelium (Fig. 3). In m a n y cases the serum proteins in the capillaries in which the bacteria-containing cells were observed appeared highly condensed. Discussion
The two strains of A. pyogenes cross-reacted strongly in crossed immunoelectrophoresis, as might be expected from the observation that A. pyogenes is a serologically homogeneous species (Ryff and Browne, 1954; Sorensen, 1974). The development and application of a PAP-technique for the demonstration of A. pyogenes in tissues was specific on murine material at a primary antiserum dilution of 1 in 256 (plateau end-point). However, on bovine tissues some non-specific staining was observed at this dilution, but by
B o v i n e P I M s a n d A. p y o g e n e s
Fig. 2.
203
Part of a PIM lying between erythrocytes (E) in a lung capillary from a cow with pyaemia caused by A. pyogenes. Two bacteria can be seen in a lysosome (arrow) in the PIM. Note the presence of micropinocytosis vermiformis (M). EM. Bar ~ 1 gm.
a further four-fold dilution the primary antiserum was also rendered suitable for use on bovine tissues. The PAP-positive contents of macrophages in chronic cardiac lesions were usually not stained by the Gram method, and on the heart valves only some of the extracellular bacteria appeared Gram-positive, the remainder being Gram-negative. The cells ofA. pyogenes, especially when ageing, tend to become Gram-variable or negative (Sorensen, 1974; Gu6rin-Faubl~e et al., 1992). From their morphology, the bacteria-containing cells were identified as PIMs (Rybicka et al., 1974; Warner et al., 1986). The light and electron microscopical findings demonstrated that PIMs played a part in the uptake of A. pyogenes from the blood. The uptake of bacteria and particles by PIMs is followed by the release of a variety ofvasoactive and inflammatory substances, including thromboxanes and interleukin-1 (IL-1) (Miyamoto et al., 1987; Chitko-McKown et al., 1992). The thromboxanes are important mediators of lung microvascular injury, causing increased vascular pressure, increased neutrophil adherence to the endothelium, and pulmonary oedema (Malik et al., 1985). IL-1, a potent cytokine, has a central role in the inflammatory response. Among the activities ascribed to IL-1 are neutrophil activation, increased endothelial procoagulant activity, and increased adherence of neutrophils to the endothelium (Dinarello, 1992). Therefore, it is reasonable to assume that the accompanying inflammatory lung lesions in the present case
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P.S. Leifsson et al.
Fig. 3. Part of a PIM in a lung capillary. The cell contains large phagolysosomesin which a number of more or less decayed bacteria and bacterial walls can be seen. At both sides of the figure the alveoli and the alveolar epithelium (A) are shown. The PIM is in intimate contact with the endothelium (E) at the right side. Nucleus (N). EM. Bar = 1gin.
were caused b y the release o f vasoactive a n d i n f l a m m a t o r y substances f r o m the activated PIMs. In conclusion, b y PAP staining a n d electron m i c r o s c o p y it was d e m o n s t r a t e d that A. pyogenes cells m a y be taken up b y bovine P I M s . As well as clearing A. pyogenes f r o m the blood, the activated P I M s also a p p e a r e d to m e d i a t e thrombosis, infarction a n d inflammation, resulting in abscessation. T h u s , P I M s w o u l d seem to have an essential role in the initial stages o f the embolic p n e u m o n i a that occurs frequently in cattle with A. pyogenes p y a e m i a .
Acknowledgments This study was supported by the Icelandic Association of Dairy Farmers.
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Received, September 19th, 1994] Accepted, November 2nd, 1994 J