Pastewella haemolytica A1 and Bovine Respiratory Disease: Pathogenesis Laurence 0. Whiteley, DVM, PhD, Samuel K. Maheswaran, BVSc, PhD, Douglas J. Weiss, DVM, PhD, Trevor R. Ames, DVM, MS, and Mathur S. Kannan, BVSc, PhD

The severe fibrinonecrotic pneumonia associated with pneumonic pasteurellosis usually results from colonization of the lower respiratory tract by Pusteurellu huemolyticu biotype A, serotype l(A1). Despite recent research efforts, the authors lack a detailed understanding of the interactions and host response to P. huemolyticu in the respiratory tract. The authors hypothesize that management and environmental stress factors or viral infection alters the upper respiratory tract (URT) epithelium allowing P. huemolyticu to colonize the epithelium. Once the URT is colonized, large numbers of organisms enter the lung where they interact with alveolar macrophages. Endotoxin, released from the bacteria, crosses the alveolar wall where it activates pulmonary intravascular macrophages, endothelium, neutrophils, lymphocytes, platelets, complement, and Hageman factor leading to complex interactions of cells and mediators. It is the progression of this inflammatory response with neutrophil influx that is ultimately responsible for the pulmonary injury. Leukotoxin is a major virulence factor of P. haemolyiicu that allows it to survive by destroying phagocytic cells. At subcytolytic concentrations it may also enhance the inflammatory response by activating cells to produce mediators and release reactive oxygen metabolites and proteases. (Journal of Veterinary Internal Medicine 1992; 6:ll-22)

Evidence for Pasteurella haemolytica A1 as the Primary Pathogen

Shipping fever or bovine pneumonic pasteurellosis was first described in the United States in 1915 and in the United Kingdom in 1925.' It currently causes upwards of one billion dollars in annual losses to the beef cattle industry in North America' which is greater than all other diseases combined. It is believed to be caused by the interaction of stressful management practices and/or viruses and bacteria.' The etiology, epidemiology, and lesions of this disease have been defined and it is generally accepted that the primary agent responsible for producing the fibrinous pneumonia seen in this disease is P. haemolyticu biotype A, serotype l(Al).'-'' The disease

From the Department of Veterinary Pathobiology (Whiteley, Maheswaran, Weiss), Department of Veterinary Biology (Kannan), and Department ofclinical and Population Sciences (Ames), College ofvetennary Medicine, University of Minnesota, St. Paul, Minnesota. Supported by USDA Special Research Grant #CSRS88-34116-4095. Dr. Whiteley's current address is the Chemical Industry Institute of Toxicology, P.O. Box 12137, 6 Davis Drive, Research Triangle Park, NC 27709. Request reprints: Trevor R. Ames, DVM, MS, Department of Clinical and Population Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55 108.

has been reproduced experimentally in calves by transthoracic or intratracheal administration of P. huemolytica A 1 alone.*.'* Despite recent research efforts, we lack a detailed understanding of the interaction and host response to P. huernolyticu A1 in the ruminant respiratory tract. This article reviews the information presently available on the interaction of this bacteria with the respiratory tract and discusses possible pathogenic mechanisms. We will not attempt to review the extensive literature on viral-bacterial synergism as it relates to pulmonary bacterial infections. In addition, we will not discuss the effect of stress on immune function and its effect on the susceptibility of cattle to shipping fever. However, the reader is referred to several articles on these s ~ b j e c t s . ~ , ~ ~ ~ * ' ~ - ' ' Several observations point to the central role that P. haemolyticu A1 has in bovine shipping fever. In clinically healthy cattle, P. huemolyticu are present in low numbers in the nasal passages and those that are isolated are predominantly biotype A serotype 2 (A2) which is rarely associated with shipping Exposure of healthy cattle to stressful agents such as viral infection, change in management practices (marketing,transportation and processing), and change in environmental (heat, cold) conditions, leads to an explosive growth and selective colonization by P. huemolyticu A1 in the upper

12

WHITELEY ET AL.

respiratory tract (URT).10.'1~18 However, the colonization rate of P. multocida does not change in these animals. In another study of twelve healthy cattle herds, repeated nasal cultures found wide-spread colonization of the URT by P. haemolytica and P. multocida although isolation from specific animals was sporadic." Pasteurella haemolytica was isolated postmortem from one or more selected areas of the nasal cavity in spite of negative antemortem culture^.'^ This suggested that in healthy cattle recurrent or reemergent colonization of the URT could occur. This recolonization has been shown to be brought about by management or environment-related stress, natural viral infection,'0.1','8,20 or experimental infection with either infectious bovine rhinotracheitis (IBR) virus or parainfluenza 3 (PI3)virus.21 These same authors showed that viral infection even in the presence of serum and nasal antibodies to P. haemolytica allowed the colonization of the URT. Once extensive colonization of the URT has occurred, bacteria can be cultured from droplet nuclei in tracheal air.22 Factors Favoring Adhesion and Colonization of the URT

Role of Mucociliary Clearance and Fibronectin Bacterial adhesion and colonization is the first step in many bacterial diseases. Much work has been done on this subject as it relates to Escherichia coli colonization in the gut and urinary tract, and Pseudomonas aeruginosa in the URT of humans.23 However, surprisingly little information is available on the mechanisms of adhesion and colonization of the bovine URT by P. haemolytica. Two major alterations that lead to colonization of the URT by gram-negative bacteria are alterations in the mucociliary apparatus24and loss of fibronectin an adhesive gly~oprotein,~' fro q epithelial cell surfaces. Loss of fibronectin, which exposes receptors on cells and permits binding of gram-negative bacteria, appears to be a key event favoring colonization of the human URT.25,26Gram-positive bacteria preferentially bind to fibronectin so the loss of this protein will decrease the level of the competing bacterial flora. Viral infections of the URT, stress and bacterial products all may have a role in altering the mucociliary apparatus and the amount of cell surface associated fibronectin. The most obvious effect of viral infection is the direct killing of epithelial cells leading to inflammation and the formation of an exudate containing iron and serum which is likely to be an excellent bacterial growth medium.27Gram-negative bacteria preferentially colonize desquamated epithelial cells27as well as immature regenerative epithelium both of which have low amounts of surface fibr~nectin.~~.~' Noncytolytic viral infections also decrease surface fibronectin-perhaps due to altered cell metabolism and increased amounts of cell surface proteases.26 Influenza virus-infected epithelial cells have increased bacterial adherence due to the ex-

Journal of Veterinary Internal Medicine

pression of viral associated receptors for ba~teria.~~-~O The effects of viruses on the efficiency of mucociliary clearance may be direct, by killing of the ciliated cells, or indirect, by altering cell secretions and ciliated cell function. Virus-altered epithelium produces more serous secretions thus decreasing the efficiency of transport of the mucus blanket by cilia.24Inflammatory mediators have been suspected of decreasing ciliary function and altering mucus quality." Leukotriene C4 (LTC4)appears to be the mediator responsible for these effect^.^' Virus-infected cells have also been shown to have decreased ciliary function. IBR, P13,or bovine virus diarrhea (BVD) viruses readily destroyed ciliary activity in bovine tracheal ring cultures.32In contrast, bovine respiratory syncytial virus (BRSV), did not destroy ciliary activity.32

Role of Stress Another factor in bovine respiratory disease is stress. Management-related stressorslo~l (transportation over long distances, crowding and comingling of animals of different sources, marketing, starvation, dehydration, change in diet, etc) and environmental stressors (abrupt change in climate)," are known to cause an increase in colonization rate of the URT of cattle by P. haemolytica Al. There is little direct information in cattle on how stress alters the URT and allows colonization. In human beings, stress alters the amount of fibronectin on the epithelial surface. After surgery, human patients have decreased amounts of fibronectin on URT epithelial surfaces, increased salivary protease concentrations, increased concanavalin A (Con. A) binding and increased susceptibility to colonization by gram-negative bacteria.25333,34 Therefore, it appears that the protease concentration in secretions can regulate the amount of epithelial cell-associated fibronectin and thus the amount of gram-negative bacterial colonization. Additionally, unstressed patients with larger amounts of Con. A binding before surgery, had more P. aeruginosa colonization during the post operative recovery period. This suggests a possible genetic predisposition to colonization of the URT by gram-negative bacteria, through increased expression of bacterial receptors. The effect of stress on mucus quality is unknown, however, adrenergic stimulation of respiratory mucus glands increases the fluidity of secretions in some animal species, but not others.35Increased fluid secretion would in turn be expected to decrease the effective movement of mucus by cilia.

Role of Bacterial Products in Adherence ofP. haemolytica Pasteurella haemolytica also produces neuraminidase and neutral protease that may directly alter the microenvironment and enhance its ability to colonize the URT.36-38Neuraminidase is a virulence factor asso-

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. NO. 1. 1992

PASTEURELLA HAEMOLYTlCA A1 AND BOVINE RESPIRATORY DISEASE

13

ciated with several gram-negative and gram-positivebacsis surrounded by a zone of bacteria and degenerating In an in vitro study, P. haemolytica A1 proswirling inflammatory cells. These classic pulmonary leduced larger amounts of this enzyme than A2.37Neursions have been documented in both the naturally occuraminidase has been shown to decrease the gel-forming ring and experimentally induced disease. However, in ~ ~ ~ capacity and viscosity of bovine respiratory ~ U C U S . ~the majority of these studies, the lesions have only been This may lead to decreased clearance efficiency of the characterized at the light microscopic level after there mucocilary apparatus and permit bacteria to penetrate has been extensive lesion development. the mucus blanket and approach the epithelium. Neuraminidase may also increase bacterial adherence by Early Events in the Genesis of the Pulmonary Lesions cleaving sialic acid residues on the cell surface.40 The cleavage of sialic acid from cell surfaceswill also decrease Role of Diferent Cell Types the net negative charge on the epithelial cell and thus More recently we4' and other^^^-'^ have begun to characallow closer approximation of the negativelycharged bacterize the genesis of the pneumonic lesions within the terial surface to the epithelial cell membrane and also first 24 hours after P. haemolytica A 1 inoculation. These permit increased adsorption on cell surfaces of cytotoxic studies have shown that neutrophils enter the lung4' fa~tors.~' However, the treatment of human epithelial within the first few hours after bacterial inoculation and cells with neuraminidase did not increase the adherence that neutrophil depletion ameliorates the pulmonary inof P. aeruginosa.2s Bacterial proteases may facilitatethe adherence of bacjury and the pathophysiologic alterations that occur in teria to the cell surface. The majority of bacterial prothe intact anima1.49.5'However, in another study, neutroteases examined have the ability to cleave fibronectin phi1 depletion did not completely abrogate pulmonary from the cell surface in ~ i t r o thus , ~ ~exposing surface lesions suggesting that other cells or bacterial virulence receptors. One of us (SKM-personal communication) factors may be involved in causing pulmonary injury.s3 has observed that brief trypsinization of bovine nasal epiWe have also observed that endothelial and alveolar epithelium leads to increased adherence of P. haemolytica thelial damage occurs immediately beneath sites of neuA 1. Bacterial proteases may also specifically cleave and trophil atta~hment.~' These changes were characterized inactivate secretory immunoglobulin by endothelial disruption and attenuation and alveolar Gram-positive bacteria contain lipoteichoic acid on epithelial cell swelling. These observations agree well their cell surface that function as an adhesin.22This molewith other experimental studies in which neutrophil decule is amphipathic, (a molecule that has both lipid and pletion has been shown to protect animals from acute water soluble components) which may allow it to interinflammatory It has also been suggested, based act with cell membrane receptor. Lipopolysaccharide in on morphologic and histochemical observations, that the outer membrane of gram-negative bacteria is also an the origin of the smudged or swirling monocytoid cells amphipathic molecule and thus may play a key role in seen in shipping fever in cattle" and Actinobacillus gram-negative adhesion to epithelium. pleur~pneumoniad~ in swine are primarily neutrophils Many gram-negative bacteria contain fimbriae or pili and not macrophages. If neutrophils are the primary effector cells of tissue that are involved in colonization of mucosal ~urfaces.4~ Fimbriae are composed of proteins that act as lectins in injury, then what factors are responsible for facilitating their migration into the lung? We hypothesize that pulrecognizing specific cell surface receptors.4345Pasteurella haemolytica A1 produces two types of fimbriae, a monary macrophages play a central role in mediating large, rigid nonflexible structure of 12 nm diameter, and the inflammatory response in the lung. There are several a small, thin flexible structure of 5 nm diameter.46,47 The different kinds of macrophages in the lung including alcapacity of other serotypes of P. haemolytica to produce veolar, interstitial, and intravascular macrophages. We fimbriae is unknown. The differential expression of fimhave observed early ultrastructural changes that suggest briae may explain the selective URT colonization of P. that both pulmonary intravascular macrophages (PIM) haemolytica A 1 after cattle are exposed to stressful manand alveolar macrophages (AMO) play a significant role agement practices, and thus explain the predominant in mediating the inflammatory re~ponse.~' In the alveorole of A 1 in shipping fever. lus, fibrin and neutrophils are often aggregated around We hypothesize that once P. haemolytica A1 coloAMO. In the capillary bed, platelet aggregation, fibrin nizes the URT it can gain entry to the lung via aspiration formation, and neutrophil and lymphocyte sequestraof droplet nuclei, colonized desquamated epithelial cells tion all appear to occur in close association with PIM. Endothelial vacuolization also occurs in association with or pharyngeal secretions. When large numbers of rapidly PIM. The PIM is highly phagocytic and was observed to growing bacteria enter the lung they produce a devastating anterior ventral pneumonia, characterized by exteningest and degrade neutrophils, platelets, red blood cells, sive fibrin exudation, neutrophil and macrophage inand fibrin. These alterations are present 4 hours postinflux, capillary thrombosis, and foci of coagulation necrofection, the earliest time point examined. The central

14

WHITELEY ET AL.

Journal of Veterinary Internal Medicine

some reports indicate that leukotoxin may act by forming pores in the cell membrane which permit the influx of calcium into ~ e l l s . ~Using ' - ~ ~ immunohistochemical techniques, on pneumonic lung sections, we have shown that leukotoxin specifically reacts with leukocytes in the lung.74Furthermore, the toxin was present in the inflammatory exudate and bound to the membranes of degenerating inflammatory cells. In other studies, alveolar macrophages, treated with cytochalasin B to inhibit phagocytosis, were equally as sensitive to leukotoxin as untreated controls.62This suggests that leukotoxin does not have to be internalized to produce its lytic effect. In Cellular and Humoral Reactions this same study, cooling the culture medium to 22" and in the Pathogenic Process 4°C inhibited the cytotoxic effect of leukotoxin on alveolar macrophages. A recent report has shown extenRole of Leukotoxin sive homology between the DNA sequence of the leukotoxin genes and the genes coding for the alpha-hemolyThe cytotoxicity of P. haemolytica for bovine leukocytes sin of E. coli.63,75 The alpha-hemolysin is known to be a was first reported in 1978.56This toxic factor termed member of a family of cytolytic bacterial toxins that act leukotoxin, has been shown to be specific for ruminant as membrane calcium pore forming proteins.76The lytic neutrophils, monocytes/macrophages, and lymphoeffect of leukotoxin appears to be dependent on the presc y t e ~ . Leukotoxin ~ ~ - ~ ~ is a heat-labile protein exotoxin, ' - ~ ~ influx into that is oxygen-stable, non-dialyzable, non-hemolytic, ence of calcium in the m e d i ~ m . ~Calcium 1) ~activation of phospholipases .~~ water soluble, and is produced by P. haemolytica only cells may i n d ~ c e : ~ during the logarithmic phase of growth.6G62The genes that release platelet activating factor and arachidonic that code for the synthesis and secretion of this leuko- acid with subsequent formation of chemotactic and vatoxin have recently been cloned.63Leukotoxin has a mo- soactive lipids; 2) activation of the respiratory burst and lecular weight of 101- 105 kDa when determined by so- degranulation; and 3) induction of the proinflammatory dium dodecyl sulfate-polyacrylamidegel electrophoretic cytokines interleukin 1 (IL-1) and tumor necrosis factor a n a l y s i ~ .However, ~ ~ , ~ using size exclusion chromatogra- (TNF). If uncontrolled calcium influx continues, cell phy or ultrafiltration it appears that in its natural envi- membranes loose their integrity as a result of continued ronment the leukotoxin exists in a multimeric state with phospholipase degradation. There is evidence that calmolecular weights of 150-300 kDa or greater.60,62,65 All cium influx leads to cell activation followed by cell 15 serotypes of P. haemolytica produce l e u k ~ t o x i n . ~ ~death. ,~~ Bovine neutrophils and alveolar macrophages, when It is highly immunogenic and antiserum raised to the 105 kDa protein neutralizes its leukotoxic activity.64 exposed to logarithmic growth phase P. haemolytica or Cattle with high leukotoxin antibody titers have higher bacterial culture supernate, undergo an initial respirasurvival rates in natural and experimental cases of pneu- tory burst followed by a precipitous decrease in oxidative monic pasteurellosis than animals with low antibody metabolism as measured by luminal dependent chemilu~ , ~ ~ - ~heat ~ killed P. haemolytica titers5 The morphologic effects of this toxin on neutro- m i n e s ~ e n c e . ~However, phils and macrophages in vitro have been de- and live P. multocida caused a more gradual rise and s ~ r i b e d . ~These , ~ ~ studies , ~ ~ show that neutrophils and sustained respiratory burst. These results suggest that macrophages respond to leukotoxin by cell swelling, or- leukotoxin is responsible for the observed respiratory ganelle and cytoplasmic membrane disruption and chro- burst pattern. More recently, data from two laboratoshowed that the leukotoxin suppressed the neumatolysis. The same changes occur in alveolar macro- ries82,83 phages when leukotoxin is instilled into the lung.69Mac- trophil respiratory burst induced by known agonists.Furrophages are more resistant than neutrophils to the lytic thermore, leukotoxin does not directly stimulate a respibut causes effect of leukotoxin, and alveolar macrophages from ratory burst in bovine n e u t r ~p h i l s , ~~ adult cattle are more resistant than alveolar macro- degranulation of lysosomes.82 The full spectrum of effects of leukotoxin on leukophages from calves less than 16 weeks of age.7oWe have also observed that peripheral blood neutrophils taken cytes is unresolved. Subcytolytic concentrations of leufrom different animals, show varied sensitivity to cell kotoxin have been shown to decrease the respiratory damage by leukotoxin (SKM-personalcommunication). burst capabilities of neutrophils when subsequently exThese variations in sensitivity may arise from regulation posed to opsonized bacterias0 and reduce chemotactic factor production of macrophages in a reversible manof receptor expression on cell surfaces. The precise mechanisms by which leukotoxin causes ner.84However, subcytolytic concentrations did not inthese morphologic changes are unknown. However, hibit the ability of alveolar macrophages to kill virus-in-

role that pulmonary macrophage may have in orchestrating the inflammatory process is discussed later. These observations suggest that there are dynamic interactions occumng between pulmonary macrophages, parenchymal cells, neutrophils, lymphocytes, platelets and the coagulation cascade which are initiated by pathogenic factors from P. haemolytica (most likely leukotoxin and endotoxin). These pathogenic factors, and the cellular and humoral events they initiate in the lung will be the focus of the remainder of this discussion.

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PASTEURELLA HAEMOLYTCA A1 AND BOVINE RESPIRATORY DISEASE

fected epithelial cellsE4Some of the conflicting results may be due to the fact that all leukotoxin preparations described to date are contaminated with endotoxin. Role of Endotoxin Pasteurella haemolytica also produces endotoxin or lipopolysaccharide (LPS). It is estimated that 12-25% of the bacteria is composed of LPSE5yE6 and that the majority of the LPS is of the smooth type.87Endotoxin interactswith numerous cell types and humoral mediator systems resulting in a plethora of pathophysiologic alteration^.^^.^^ Endotoxin can readily cross the alveolar wall either from the air or blood and interact with cells and humoral mediators. Using immunohistochemical techniques, we have shown that endotoxin is released into the inflammatory exudate and that it is localized: 1) in neutrophils, in the alveolus, interstitiurn, and capillary lumen; 2) in intravascular, interstitial and alveolar macrophages; 3) in enSimilar dothelial cells; and 4) on epithelial cell s~rfaces.'~ results were reported when endotoxin is administered to rats intraven~usly.~"-~~ We have also shown that killed P. haemolytica A1,48 given intratracheally, or purified LPS from P. haemolytica A 169given intrabronchially, is capable of causing neutrophil and fibrin exudation, pulmonary edema, and platelet and neutrophil aggregation in capillaries. Similar changes are also observed in sheep given endotoxinintrav e n o ~ s or ~ ~intrabr~nchial:~ ,~~ and in hamster^:^,^^ guinea pigs,97,98 rats99and rabbits,loo given endotoxin by inhalation or intrabronchial deposition. The mechanisms of endotoxin action at the cellu1ar'Ol-l l3 and whole animal levelsE7~8E~10'~' 1471 l5 have been extensively studied and are complex. The lipid A component of LPS has the capacity to activate serine proteasedo6and thus can activate Hageman factor, and the first component of the classical complement pathway. The polysaccharide component of LPS can activate the alternate pathway of complement.88,'06 The activation of Hageman factor leads to activation of the coagulation cascade, kinin system, plasminogen activator, and complement.88These systems are capable of producing a multitude of proinflammatory and cell activating compounds.8E The interaction of endotoxin with cells leads to cell activation and/or death. Endotoxin interacts with leukocytes, cells of the mononuclear phagocyte system, endothelium, hepatocytes, adrenal cortical cells, red blood cells, and platelets. In these cells, LPS localizes on the cell membrane, and/or in the cytosol, mitochondria, lysosomes, endoplasmic reticulum, and the nuc1eus.103~105~108~1 16-'22 Lipopolysaccharide may enter these cells by interacting with a specific receptor via the core polysaccharide or by insertion into the cell membranes via the lipid A portion of the molecule.'05-'0EIn rats, peritoneal macrophages interact with endotoxin via spe-

15

cific receptors, but Kupffer cells lack LPS receptors and internalize LPS via pinocytosis.123 Lipopolysaccharide also binds to low- and high-density lipoproteins in plasma and a variety of cell types may ingest LPS via receptors for these p r ~ t e i n s . ' ~ ~ ~ ' ~ ~ The precise mechanisms by which endotoxin produces metabolic alterationsleading to cell activation and cell death are not clear. Proposed mechanisms include increased cytosolic calcium, mitochondrial injury, production of intracellular free radicals, and activation of serine proteases. Lipopolysaccharide may interact with the cell membrane or endoplasmic reticulum to allow calcium entry and the resultant activation of metabolic pathways.'02-104In addition to altering the membrane permeability to calcium, LPS may directly interact with the mitochondria1 outer membrane and destroy the membrane proton gradient leading to loss of oxidative glycolysis.'05This may in turn lead to anaerobic glycolysis, production of lactic acid and oxygen radicals, and lysosomal membrane breakdown leading to cell death. Intracellular free radical generation may also be important in LPS-induced cell injury because cell membrane penetrating free radical scavengers are capable of preventing cell death."' One author also suggests that the lipid A moiety of LPS can directly activate serine proteases in the cell membrane and thus cause cell activation.lo6 Bovine pulmonary artery endothelium is sensitive to the stimulatory and toxic effects of endotoxin.'26 The exposure of endothelial cultures to LPS causes dose dependent shape changes, cell retraction, increased cell membrane permeability and pyknosis.126~127 These changes are accompanied by decreased synthesis of DNA, RNA and protein.'26Endotoxin or its lipid A derivative activatesbovine pulmonary endothelial cell protein kinase C and increases phosphoinositol turnover.'26 Activated endothelial cells produce a wide variety of proinflammatory and procoagulant mediators including IL- 1, platelet activating factor (PAF), prostacyclin (PGI), prostaglandin E, (PGE,), tissue factor, and plasminogen activator inhibitor activity.126-129 The direct effect of endotoxin on alveolar epithelial cells is unknown. However, 18 hours after intratracheal deposition of E. coli-derived endotoxin to rats morphologic alterations in type I1 cells and release of alkaline phosphatase into bronchoalveolar lavage fluid suggests epithelial damage.99In calves given P. haemolytica A1 endotoxin intrabronchially and examined four hours later, we demonstrated epithelial damage only at sites of inflammati~n.~~ This suggests that damage to epithelial cells is secondary to inflammation. Type I1 cells have recently been shown to release arachidonic acid when exposed to calcium ionophore, and to metabolize it primarily to PGE2130which is known to down regulate leukocyte and macrophage f~nction.'~' This may serve as a protective response for the epithelial cells, allowing them

16

WHITELEY ET AL.

to inhibit the cellular inflammatory response when they are injured. Alveolar macrophages and PIM'32-'33can directly interact with endotoxin entering the lung via the airway or blood. This interaction leads to activation of alveolar and blood macrophages both directly105and indirectly via complement activation.88.'06 Endotoxin, at high doses, may also be toxic to alveolar macro phage^.'^^ These macrophages are able to produce a wide array of p r ~ i n f l a m m a t o r y , ' ~ ~pro~oagulant,'~'-'~~ -'~ and toxic oxygen radicals and pro tease^.'^^,'^ Of particular interest are the cytokines (IL-1, TNF) and the lipid mediators (PAF, LTB,, LTC, and LTD,), which are centrally involved in the inflammatory response. The effects of these mediators are overlapping. Both IL- 1 and TNF are chemotactic for neutrophils, monocytes and lymphocytes and they activate neutrophils to produce toxic oxygen radicals and to degran~1ate.I~~ More recent studies using highly purified IL-1 indicate that it is not chemotactic but does stimulate neutrophil degranulation and respiratory b ~ r s t . ' ~ ~ , A' ~6"kD ' ~a~polypeptide, released from human endotoxin-stimulated monocytes, has recently been identified, which is chemotactic for neutrophils, and causes degranulation and stimulation of respiratory burst.'49This factor has been called by a variety of names: monocyte-derived neutrophil chemotactic factor, neutrophil activating protein- I , neutrophil activatInterleukin- I and ing factor, and interle~kin-8.'~~,'~~*'~~ TNF can stimulate human monocytes to produce monocyte-derived neutrophil chemotactic factor (IL-8).I5' Interleukin- 1 and TNF also activate endothelial cells and induce procoagulant activity on their surface.'52 These cytokines also increase the expression of adhesion molecules such as intercellular adhesion molecule- 1 (ICAM-I) on endothelial cell surfaces and the family of CD 18 adhesion molecules on neutrophil cell surfaces.'45 These adhesion molecules are essential for neutrophilendothelial adhesion and neutrophil migration into the alveolus.'45Interleukin-I and TNF can act in an autocrine manner to activate macro phage^.'^^ They also increase endothelial susceptibility to neutrophil induced damage.'53 The inflammatory mediators LTB, and PAF produce effects similar to IL-I and TNF. They are chemotactic for, and induce oxidative metabolism and aggregation of n e u t r ~ p h i l sThey . ~ ~ increase the expression of adhesion molecules on neutrophils. Leukotriene B, may also induce increased adhesiveness of the endothelial cell surface.'54 Both PAF and LTB, can activate endothelial cells and increase their sensitivity to neutrophil inj ~ r y . ' ~Leukotrienes ,'~~ C, and D, increase capillary permeability and cause bronchoconstriction.30They also induce PAF production by endothelial cells and increase endothelial adhesiveness for neutrophils. Recently PIM and AM0 from swine have been shown to metabolize arachidonic acid and produce thrombox-

Journal of Veterinary Internal Medicine

ane A, (TxA,), LTB,, LTC, and other lipoxygenase met a b o l i t e ~ . 'This ~ ~ study showed that PIM are capable of producing larger quantities of these products than AMO. This is particularly interesting in light of the fact that A M 0 can metabolize arachidonic acid released from epithelial cells and produce LTB, and 12-hydroxyeicosatetraeonic acid, thus enhancing the production of chemotactic factors for n e ~ t r o p h i l s . The ' ~ ~ possibility of intercellular exchange of mediators between macrophages and parenchymal cells of the lung poses interesting possibilities for the regulation of the inflammatory response in the lung. Endotoxin does not directly stimulate neutrophils, but in picogram concentrations can prime neutrophils for increased oxidative metabolism and lysosomal enzyme release in response to a second a g ~ n i s t . ' ~ ~This - ' ~ second ' agonist can be one of several different compounds such as C5,, bacterial chemotactic factors, leukotrienes, and possibly c y t o k i n e ~ . ' ~Thus, ~ . ' ~ endotoxin cannot only prime neutrophils but can generate the secondary stimulus for their activation. These activated neutrophils are responsible for tissue injury by releasing both toxic oxygen radicals and lysosomal enzymes. They can further exacerbate the inflammatory response by producing LTB,, PAF30,'54and possibly IL- I .I6, Endotoxin also activates l y r n p h o c y t e ~ . ~Activated ~.'~ T-lymphocytes produce gamma-interferon (gINF)'04 and TNF'45which are capable of activating macrophages thus leading to augmentation of the inflammatory response. Lymphocyte-derived TNF activates neutrophil oxidative metabolism. Also, the induction of procoagulant activity in macrophages is lymphocyte-dependent. 164 The role of lymphocytes in the acute inflammatory response in bovine shipping fever is speculative. However, some interesting observations implicate their involvement. There is a marked decrease in circulating l y r n p h o c y t e ~ with ~ . ~ ~sequestration in the lung48a few hours after experimental inoculation of P. huemolyticu A I. Also, lymphocyte depletion of sheep, by thoracic duct drainage, markedly reduces the early pulmonary injury after intravenous endotoxin admini~tration.'~~ Endotoxin can directly or indirectly activate platel e t ~ . ~ ~ ,These ' ~ ' , 'activated ~~ platelets release numerous products that augment the inflammatory cascade, including PAF, TxA,, arachidonic acid, endoperoxides, and 12-lipoxygenase metabolites.'66 Endoperoxides and arachidonic acid can be metabolized by other cells such as macrophages, neutrophils and endothelial cells leading to augmented production of lipid mediators. However, it is unlikely that platelets have a direct role in the pulmonary response to endotoxin because platelet depletion of sheep does not alter the pulmonary response to endotoxin ~ha1lenge.I~~ It is apparent that the proliferation of P. huemolyticu A 1 in the lung leads to the activation of resident pulmo-

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PASTEURELLA HAEMOLYTCA A1 AND BOVINE RESPIRATORY DISEASE

nary cells and leukocytes and generation of numerous inflammatory mediators. The question then arises as to which factors and cells are central to initiating the inflammatory response. Pulmonary macrophages (PIM and AMO) are uniquely situated to interact with infectious agents or toxic compounds entering the lung. In addition, it is becoming increasingly evident that the macrophage products IL- 1, TNF and IL-8 are essential for the generation of a sustained inflammatory response with Several lines of evidence neutrophil influx.'34J47~'49J50~'62 support this hypothesis: a) the accumulation of neutrophils in the lungs of mice after the inhalation of LPS is dependent on metabolically active alveolar macrophages;'68b) administration of antimacrophage antibodies in rat peritoneal cavity prevents neutrophil accumulation in response to locally deposited e n d ~ t o x i n ;and '~~ c) decomplementation, both systemic and intraalveolar, does not affect neutrophil accumulation in the lung after LPS a e r o s ~ l i z a t i o n . ~ ~0 J ne ~ ' Jgroup ~ ~ of investigators demonstrated that the influx of neutrophils in response to subcutaneously deposited endotoxin is dependent on protein synthesis, and that cross-tachyphylaxis between endotoxin and IL-1 could be demonstrated for neutrophil recruitment but did not occur between endotoxin and LTB, or activated ~ o m p l e m e n t . ' ~ ' -On ' ~ ~a molar basis, endotoxin is equipotent with IL-1 and 1000 times more potent than PAF, LTB,, and C5ain inducing neutrophil i n f l u ~ . ' ~ ~Antibodies * ' ~ ~ * ' ~to ~ TNF are able to block the lethal effects of endotoxin in mice'45and antibodies to TNF and IL- 1 prevent neutrophil-induced injury to endothelial cells.153 The ability of IL- 1, TNF, and IG8 to induce neutrophil influx into an inflammatory site is due to their ability to directly induce chemotaxis (TNF, IL-8) and indirectly (IL- 1, TNF) by inducing the production, by macrophages, of lipid chemotaxins such as PAF and LTB,. However, the ability of IL-1 and TNF to induce the expression of adhesion molecules on both endothelial and neutrophil cell surfaces is essential in permitting neutrophil migration into an inflammatory ~ i t e . ' ~ ~ ,F' ' igure ~~,'~' 1 illustrates some of the interactions that may be taking place in the lung in response to P. haemolytica. Therapeutic Strategies to Control Acute Lung Injury The major approach for treating pneumonic pasteurellosis has been antibiotic therapy. Until recently, the mechanisms responsible for the acute lung injury were poorly understood, and few pharmacologic agents were available to limit lesion development. Several compounds are available that may modulate the inflammatory response. In addition, a clearer understanding of the disease process may facilitate more appropriate use of existing compounds. Both new and existing agents may either act selectively against specific mediators or groups of mediators or act against multiple mechanisms of injury.

17

Cyclooxygenase inhibitors are widely used compounds that inhibit synthesis of cyclooxygenase catalyzed products. Flunixin meglumine, phenylbutazone, and aspirin are examples of compounds that are currently in use in veterinary medicine. Flunixin meglumine has been demonstrated to alleviate many of the endotoxin-induced metabolic, hemodynamic, and clinical changes in the h ~ r s e . ' ~Indomethacin, ~,'~~ ibuprofen, and other potent cyclooxygenase inhibitors have been reported to reduce lung injury due to E. cofi endotoxemia or bacteremia in and goats.'78Cattle experimentally infected with P. haemolytica and treated with flunixin meglumine and oxytetracyclinehave shown improved clinical response and decreased lung involvement compared with those treated with oxytetracycline alone.'79Other selective inhibitors of thromboxane synthesis, or antagonists for PAF are currently being developed and evaluated.l8' Hopefully, some of these will prove useful and effective and can be economically applied to pneumonic pasteurellosis. With all of these specific antagonists and inhibitors, it is essential that therapy be initiated early in the course of the disease to be effective in limiting lung injury. As mentioned previously, oxygen radicals released by PMNs may also induce toxic damage to the lungs. Free radical scavengers, such as dimelthyl sulfoxide (DMSO), if administered early in the disease process, may be effective in penetrating the cell membrane and removing harmful radicals that may cause cell death. No research to date, however, supports the use of DMSO as an effective treatment for pneumonic pasteurellosis. Other agents that would limit generation of toxic oxygen radicals would include iron chelating agents which inhibit radical reactions. Deferoxamine, an iron chelating agent, has been reported to decrease lung injury after complement activation. "' The therapeutic benefit of deferoxamine in bovine pneumonic pasteurellosis is controversial. In calves experimentally inoculated with P. haemofytica, deferoxamine mesylate administered twice daily did not attenuate the lung lesions.182However, the halflife of deferoxamine mesylate in rats is only 5 minutes. Administration of deferoxamine bound to hydroxyethyl starch, which extends the half-life to 90 minutes, was shown to attenuate the increase in total protein, albumin, alkaline phosphatase and lactic dehydrogenase in bronchoalveolar lavage fluid 4 hours after intratracheal administration of P, haemolytica. 183 Modulators with multiple modes of action for limiting lung injury would include corticosteroids. These agents have been available for use in cattle for years and have long been advocated by some practitioners for use in pneumonic pasteurellosis. The principle action of corticosteroids in controlling inflammation is their ability to inhibit phospholipase A2, a membrane enzyme whose activation triggers generation of arachidonic acid and PAF.'84,'85The clinical use of corticosteroids in human

18

Journal of Veterinary Internal Medicine

WHITELEY ET AL.

I

actiity

'I 1 1

FIG. 1. The possible interactions of P. haemolytica Al-derived endotoxin and leukotoxin with the bovine lung. Pulmonary intravascular macrophage (PIM), alveolar macrophage (AMO), interleukin-1 (IL- 1), tumor necrosis factor (TNF), gamma-interferon (gINF), arachidonic acid (AA), intercellular adhesion molecule-1 (ICAM-I), fibrin degradation products (FDP), active 5th component of complement (CSa), platelet activating factor (PAF), thromboxane A, (TXA,), prostacyclin (PGI), leukotriene B,, C,, D, (LTB,, LTC,, LTD,), interleukin-8(IL-8), platelets(P), hydroxyl radical (OH ).

.

adult respiratory distress syndrome (ARDS) remains a subject of debate. Corticosteroidsare used with the hope that high doses given at or before the onset of lung injury could slow down, arrest, or reverse the injury associated with ARDS.''' A large study on feedlot cattle with respiratory disease revealed animals receiving dexamethasone and tetracyline had a poorer response to initial treatment and had more relapses than did cattle receiving tetracyclines alone.1s6The unfavorable results found in this study probably occurred from the immunosuppressive effects of dexamethasoneoutweighingthe immediate control of lung injury. No attempt at case selection was made in this study and other causes of respiratory disease including viral agents may have been included. Further evaluation of corticosteroids given at various stages of experimentally induced pasteurellosis would be necessary before it can be concluded that they are not beneficial in the treatment of acute pneumonic pasteurellosis.

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