Kim E. Longworth, Ph.D.

Department of Surgical and Radiological Sciences, School of Veterinary Medicine University of California, Davis, CA

Received 4/11/97; Accepted 5/21/97

TABLE OF CONTENTS

1.Abstract
2.Introduction
3.Morphology and distribution
4.Physiology
4.1 Effect on pulmonary hemodynamics
4.2 Secretory and immune functions
5.Role in disease
6.Perspective
7.References

1. ABSTRACT

Pulmonary intravascular macrophages are an important part of the mononuclear phagocyte system in some species of mammals, mainly sheep and other ruminants, pigs, and horses. These cells phagocytize foreign particles, cell debris and pathogens that pass through the pulmonary circulation. Species with intravascular macrophages localize intravenously injected tracer particles and bacteria predominantly in the lung rather than the liver, and exhibit pulmonary hypertension when these cells are activated. Both in vivo and in vitro studies show that pulmonary intravascular macrophages have distinct secretory and immune capabilities. Consequently, the pulmonary intravascular macrophages play an important role in pulmonary inflammation in species that have them.

2. INTRODUCTION

Pulmonary intravascular macrophages are a distinct population of cells that permanently reside within the pulmonary capillaries of some species of mammals. Research on the physiological significance of these cells has only developed in the last 10 years. However, it is clear from this research that, in species that have them, the macrophages play an important role in the animal's response to some invading pathogens, endotoxin or foreign particles (1-7).

Since 1988, reviews (1, 2, 3, 4, 5) a monograph (6) and a book chapter (7) have focussed on various aspects of pulmonary intravascular macrophage biology. As the first publication of this topic to be presented in an electronic forum, this review will emphasize some of the key features of the anatomy and physiology of these cells that were touched upon in published reviews.

The mononuclear phagocyte system in mammals is comprised of phagocytic cells throughout the body responsible for removing particles from the circulation (8, 9). The liver has historically been regarded as the functional center of that system because of the Kupffer cells (stellate macrophages) lining its sinusoids and because of the high fraction of systemic blood flow (about 20%) that it receives. The spleen and bone marrow, with their associated macrophage populations, are secondary sites for particle clearance, mainly because their blood flows are low.

This hepatic orientation of regional phagocytosis is based on studies in typical laboratory mammals, and humans (9, 10). Early studies showed that intravenously injected foreign particles are mostly retained by the liver and spleen in rats, rabbits and dogs (11, 12). However, although these studies suggested that the lung could retain a substantial fraction of blood-borne particles in some species, this observation was largely ignored for many decades (1). More recently, the macrophages responsible for lung retention of particles were considered to be displaced Kupffer cells by Schneeberger-Keeley and Burger (13), but later were formally identified as a resident population of macrophages by Rybicka et al. (14). Later studies confirmed the pulmonary orientation of phagocytosis in sheep, pigs, cats and calves (15, 16, 17, 18, 19).

The mononuclear phagocyte system of the lung is comprised of cells in 3 compartments; airway and alveolar macrophages, interstitial macrophages and, in some species, intravascular macrophages (20) (Fig 1). All of these macrophages may derive from sequestered monocytes in the pulmonary capillaries that migrate to the respective compartments, or may multiply by mitosis in situ (9, 21). These cells act as a portion of the lungs' defense mechanism against airborne pathogens or foreign particles that infiltrate the lung airspaces. In all mammals, blood-borne pathogens or particles that infiltrate the pulmonary tissues may be attacked by interstitial macrophages. In those species with intravascular macrophages, all particles circulating in the blood can potentially be phagocytized by intravascular macrophages before entering the interstitium.

Figure 1. Mononuclear phagocytes in the lung are a dynamic system. Large numbers of monocytes pass through the pulmonary circulation and may become transiently sequestered in pulmonary capillaries. Some of these sequestered cells migrate into the interstitium or alveolar spaces, and differentiate into mature macrophages. In some species of mammals, monocytes permanently adhere to the endothelium and differentiate into mature pulmonary intravascular macrophages. Reprinted with permission from Reference 4.

3. MORPHOLOGY AND DISTRIBUTION

Rybicka et al. first identified pulmonary intravascular macrophages as a distinct population of cells in calves (14). The mononuclear cells they observed in pulmonary capillaries of normal calf lungs had all the characteristics of mature macrophages rather than circulating monocytes. Later, Winkler and Cheville (22) and Warner and co-workers (16) showed that the cells form membrane adhesion complexes with the underlying endothelial cells, supporting the concept that they are resident rather than merely circulating cells. Workers attempting to isolate the cells by washing calcium chelators through blood-free lungs have found it difficult to break down the adhesion complexes without using digestive enzymes (23, 24, 25); such tenacious attachment to the underlying endothelium suggests that these cells are resident macrophages rather than mobile or circulating cells. In addition, putative intravascular macrophages, isolated from pig lungs, form adhesion complexes with endothelial cells (23).

As are other mature macrophages, pulmonary intravascular macrophages are larger (20-80 µm) than monocytes and have larger and more extensively developed organelles (nucleus, Golgi apparatus, mitochondria, rough endoplasmic reticulum) (Figures 2 and 3). They have numerous pseudopods that allow a large portion of the cell surface to be in contact with the endothelium. Their plasma membranes, coated with a glycocalyx, have invaginations that form micropinocytosis vermiformis; these features indicate active receptor-mediated endocytosis (2). Atwal and co-workers have extensively studied the morphological function of the surface coat (26, 27, 28). Even in normal lungs, large phagosomes contain cellular debris, indicating that clearance of effete cells from the circulation is a normal function of the cells (16, 18, 22, 29, 30).

Figure 2. Electron micrograph of capillaries (Cap) surrounded by alveolar air spaces (Alv) in a lamb lung fixed by vascular perfusion. Pulmonary intravascular macrophages (PIM) are in intimate contact with the capillary endothelium by pseudopods. Reprinted with permission from Reference 48.

Figure 3. Electron micrograph of capillaries (Cap) surrounded by alveolar air spaces (Alv) in a lamb lung fixed by tracheal insufflation to preserve the vascular contents. The pulmonary intravascular macrophage (PIM) has a characteristic fuzzy coat (glycocalyx; shown by arrow) that is not apparent in lungs fixed by vascular perfusion. L, lymphocyte. Reprinted with permission from Reference 48.

In certain species, abundant macrophages have been identified histologically in pulmonary capillaries. In 1-month-old pigs, intravascular macrophages occupy 25% of the capillary lumen and are estimated to number 14 x 10³ cells per mm³ of lung parenchyma (30); in adult sheep, this fraction is 15% and the number is 8 x 10³ cells per mm³ of lung parenchyma (16). By contrast, in other species the presence of even a small population of pulmonary intravascular macrophages is disputed (4). Although Warner et al. (2) found no electron microscopic evidence of any pulmonary intravascular macrophages in rats, Niehaus (31) suggests that they do exist in rats in small numbers. In one study in humans, investigators examined biopsies of lung tissues obtained during surgery (32). Because the samples were biopsies, the blood was not removed from capillaries, a technique which improves identification of resident intravascular macrophages. The authors reported finding occasional large mononuclear cells that appeared similar to intravascular macrophages in other species. They concluded that the population of intravascular macrophages must be relatively smaller in human lungs.

4. PHYSIOLOGY

Because of this uncertainty relating to the anatomical definition of a resident phagocyte in pulmonary capillaries of fixed tissue, it is useful to use functional criteria to determine whether pulmonary intravascular macrophages are a biologically significant cell component in the normal mammalian lung. One functional criterion is whether the lung is capable of retaining a substantial fraction of blood-borne particles.

Comparative studies have examined the uptake of intravenously injected non-biological tracer particles in mammals (Figure 4) (11, 19, 33-39). The result of combining the data from these studies shows a pattern of particle retention in the lung that is related to mammalian taxonomic orders. All seven species studied in two mammalian orders, Artiodactyla and Perissodactyla, show high lung retention of particles (generally >40%). All three species in another order, Rodentia, show low (<10%) lung retention of particles. On the other hand, of the two carnivores in which more than one particle has been tested, the cat shows variable but generally high retention and the dog shows low retention.

Figure 4. Summary of data from comparative studies showing pulmonary retention of tracer particles injected into the circulation of 16 species of mammals in 6 taxonomic orders. Orders represented (from bottom to top) are Artiodactyla (sheep, bovid [cattle, calf], goat, pig, reindeer and llama); Perissodactyla (equid [pony, horse]); Carnivora (cat, dog, ferret); Lagomorpha (rabbit); Rodentia (mouse, guinea pig, rat); Primate (monkey) and; Hyracoidea (hyrax). Particles used for injection are gold colloid, iron oxide, Monastral blue, liposomes, manganese dioxide and lipid emulsion. Data from 11, 19, 33-39.

Studies using bacteria or endotoxin in paired species comparisons support this lung retention pattern. Pigs and sheep have a high lung retention of Pseudomonas bacteria while rats and dogs have a low retention (40, 41). Regardless of the significance of the distribution among orders, it is clear that in certain species the anatomically observed pulmonary intravascular macrophages are numerous enough to effect lung localization of foreign particles or cell debris in the blood. Thus, these species could be described as having a functional pulmonary intravascular macrophage population.

However, this description of experimental observations of the fate of intravenously injected particles may not reflect the degree to which, in the natural condition, pulmonary intravascular macrophages play a role in phagocytosis of particles from the circulation. In fact, most particles injected into the portal circulation in sheep are mainly taken up in the liver by Kupffer cells (42).

A relevant criterion that could be used to assign pulmonary intravascular macrophages a primary role in the mononuclear phagocyte system is whether they elicit a physiological response over and above phagocytosis when they encounter particles. The ability of the cells to effect pathophysiological changes would have a bearing on their significance in the whole body immune defense system.

Two sets of data relate to this concept, one is the pulmonary circulatory hemodynamic response when particles are phagocytized by intravascular macrophages, and the other is the cells' secretory and immune capabilities.

4.1. Effect on pulmonary hemodynamics

Intravenous injection of particles induces pulmonary hypertension in species with pulmonary intravascular macrophages (Figure 5). In sheep, calves, pigs, llamas, goats, reindeer and horses, the tracer particles Monastral blue or liposomes induce a transient (3-5 min) increase in pulmonary arterial pressure of 16-43 mm Hg, depending on the dose and species (19, 35-39, 43). The pulmonary hemodynamic response to tracer particles also occurs in sheep when particles are injected intraarterially (33, 43). The particles are not retained in the systemic circulation, and return to the pulmonary circulation via the pulmonary artery. Species without the macrophages (dogs, ferrets, rabbits and monkeys) have almost no hemodynamic response to the same particles (39). The only exception to this observation is the cat. The cat has many macrophages that phagocytize injected particles; however, intravenous injection of particles into cats does not induce pulmonary hypertension.

Figure 5. Summary of data from comparative studies showing change in pulmonary arterial pressure after injection of tracer particles into the circulation of 13 species of mammals in 5 taxonomic orders. Orders represented (from bottom to top) are Artiodactyla (sheep, calf, goat, pig, reindeer and llama); Perissodactyla (equid [pony, horse]); Carnivora (cat, dog, ferret); Lagomorpha (rabbit); Rodentia (rat); and Primate (monkey). Particles used for injection are liposomes, Monastral blue, microspheres, rabbit blood, rat blood and goat blood. Data from 19, 33, 35-39, 43.

The pattern in species-specific response is similar when foreign blood is injected. In goats, when rabbit or rat blood is injected intravenously they induce an increase in pulmonary arterial pressure of 24 and 18 mmHg, respectively (44). No increase in pressure greater than 7 mmHg occurs in rabbits injected with goat or rat blood, or rats injected with goat or rabbit blood. In addition, sheep pulmonary intravascular macrophages phagocytize platelets bound to injected anti-sheep platelet serum; this event is associated with pulmonary hypertension (45). No pulmonary hemodynamic response occurs in rats after anti-sheep platelet serum is injected.

The transient pulmonary hypertension after intravenous injection of particles is due to pulmonary venous and arterial constriction (35) . Studies in goats, ponies and sheep have demonstrated either no change or a decrease in cardiac output concurrent with pulmonary hypertension (33, 38, 46, 47). The vasoconstriction is caused by large quantities of thromboxane A₂ (a potent vasoconstrictor metabolite of cell membrane arachidonate) released into the pulmonary circulation (measured as the pulmonary arterial-systemic arterial difference in the stable metabolite thromboxane B₂) (19, 45, 46, 47) .

In lambs, the pulmonary vascular reactivity has been linked to the presence of the macrophages (46). In newborn (1-2 day old) lambs, few macrophages are present in the pulmonary capillaries (48), and the animals show no hemodynamic response to intravenous Monastral blue or liposomes. Particles are mostly retained by the liver. By two weeks, a substantial population of macrophages appears in the capillaries, the animals respond to particles with an increase in pulmonary vascular driving pressure (left atrial-pulmonary arterial pressure difference), and particles are mostly retained by the lung. The increase in driving pressure is due to an increase in the pulmonary production of thromboxane (46).

The species differences in the pulmonary hemodynamic response to tracer particles are similar to differences observed when bacteria are injected intravenously. In response to the same relative dose of Pseudomonas aeruginosa, anesthetized pigs had a 220% increase in pulmonary arterial pressure and a 300% increase in pulmonary vascular resistance within one hour, compared to no change in pressure and only a 30% increase in resistance in anesthetized dogs (40).

The presence of pulmonary intravascular macrophages causes the pulmonary circulation to be sensitive to relatively small doses of intravenously injected endotoxin. Experimental doses of endotoxin used in species that have the cells are three orders of magnitude smaller than those used in species without the cells. For example, intravenous injection of 0.03 to 1.3 micrograms per kilogram are used to induce pulmonary hypertension in sheep and horses, and changes in lymph flow and pulmonary inflammation in sheep (49, 50, 51, 52). In contrast, experimental endotoxin doses that are used to induce systemic hemodynamic changes in rats, rabbits, dogs and non-human primates are in the range of 0.1 to 5 milligrams per kilogram of endotoxin (53-56); large doses of 0.15 mg/kg in the dog (54) and 5 mg/kg in the monkey and rabbit (53) are needed to elicit any detectable change in pulmonary arterial pressure. The maximum dose used in experimental endotoxemia in humans (4 ng/kg) does not produce any increase in pulmonary arterial pressure (57).

There is, therefore, a clear link between the presence of pulmonary intravascular macrophages and the pulmonary hemodynamic response to blood-borne particles. Among the species studied, whenever the cells are present, pulmonary hypertension occurs when foreign particles are injected into the peripheral circulation. The reactive macrophages can alter pulmonary hemodynamics and potentially alter pulmonary fluid balance.

4.2. Secretory and immune functions

Some studies have examined various secretory properties and immune activities of in vivo and isolated pulmonary intravascular macrophages (3). A few studies have focused on comparisons between these cells and alveolar macrophages. Both pulmonary intravascular and alveolar macrophages isolated from pigs and incubated with arachidonic acid produced lipoxygenase and cyclooxygenase metabolites (Prostaglandin F₂ , hydroxyheptadecatrienoic acid (HHT), and 5-, 12- and 15-hydroxyeicosotetraenoic (HETE)). However, intravascular macrophages also produced thromboxane B₂, prostaglandin D₂ and prostaglandin E₂ (58, 59). When exposed to various particles and soluble stimuli (calcium ionophore A23187, asbestos, iron spheres, zymosan, lipopolysaccharide) both types of cells produced leukotriene B₄. However, there were differences in the amounts of metabolites released by the two cell populations. The authors concluded that intravascular macrophages are generally metabolically more active than alveolar macrophages (3).

A comparison of immunological functions between isolated intravascular and alveolar macrophages from pigs showed similar bactericidal and antibody-dependent cellular cytotoxic activities (60). Although intravascular macrophages were less effective at phagocytosis and non-MHC restricted cellular cytotoxicity, they had more tumoricidal activity than alveolar macrophages. Both cell populations produced similar concentrations of interleukins 1 and 2 and tumor necrosis factor alpha (60). In studies with endotoxin-stimulated intravascular and alveolar macrophages, the former produced more T-cell proliferative cytokines and the latter more tumor necrosis factor alpha and nitric oxide (61).

In vivo studies suggest that there are differences in the secretory capabilities of intravascular macrophages and Kupffer cells (4). The pulmonary circulations of species with intravascular macrophages release thromboxane into the circulation in response to injected particles (19, 44, 46, 47) . In contrast, in species without intravascular macrophages, although injected particles are taken up by the liver, there is no apparent release of thromboxane into the systemic circulation.

These studies show that pulmonary intravascular macrophages are as metabolically active as other macrophage populations, and that they have a full range of secretory and immune capabilities, apparently different from alveolar macrophages and Kupffer cells. Taken together, the data indicate that the macrophages are important in mediating pulmonary pathophysiological changes.

5. ROLE IN DISEASE

A few studies have specifically focused on the role of pulmonary intravascular macrophages in pulmonary disease. When Pseudomonas aeruginosa bacteria are intravenously injected into sheep or pigs, the pulmonary capillaries become congested with red cells, neutrophils, platelets, some lymphocytes and fibrin clumps, and intravascular macrophages (40-41, 62). Lung interstitium shows varying degrees of edema, from widening around larger vessels to intraalveolar edema with atelectasis. One hour after injection, bacteria in the lung parenchyma are contained within phagosomes of intravascular macrophages (41); by 24 hrs they are also found in neutrophils (62). These morphologic changes accompany physiological changes, such as pulmonary hypertension and delayed systemic hypotension, an increase in lung lymph flow (initially due to hydrostatic pressure and later to increased vascular permeability), hypoxemia, fever and leukopenia (40, 62).

In contrast, rats and dogs injected with Pseudomonas show almost no pulmonary morphologic changes and no features of pulmonary failure (40-41). In the rat, most bacteria are retained in the liver by Kupffer cells, and it is the hepatic sinusoids that are congested with neutrophils and platelets (40). In dogs, the hemodynamic effect of bacterial infusion is limited to systemic hypotension and decreased cardiac output (41).

Other studies on different pathologic organisms demonstrate the involvement of intravascular macrophages in lung infections. Bertram showed that after intratracheal inoculation with Haemophilus pleuropneumoniae, the relative volume of intravascular macrophages in pig lungs increased in areas of inflammation and necrosis (63). The cells matured within 24 h of inoculation, increasing in cytoplasmic volume (due to an increase in the number of organelles), and increasing intracellular adhesion to underlying capillary endothelial cells. Bertram concluded that the intravascular macrophages clear cellular and acellular debris from the blood in pneumonitis.

A similar role has been proposed for pulmonary intravascular macrophages in the pulmonary response to intratracheal inoculation of Pastuerella haemolytica in calves (64), in experimentally induced African Swine Fever (a virally-induced hemorrhagic disease) in pigs (65, 66), and in the response to spontaneously occurring Actinomyces pyogenes lesions in cattle (67).

6. PERSPECTIVE

Experimental studies suggest that pulmonary intravascular macrophages have a dominant role in the mononuclear phagocyte system of mammals in the orders Artiodactyla and Perissodactyla. These cells are involved in clearance of particles and debris from the circulation and the immune response against blood-borne and airborne pathogens. It is less clear what their importance is to animals in the natural environment. Although various hypotheses have been proposed to account for the presence of these macrophages, it is uncertain why such a substantial and distinctive population has evolved in some species and not others (4).

Much of the current research focuses on whether pulmonary intravascular macrophages are induced in humans in certain pathological conditions. Humans normally have few intravascular macrophages; foreign particles in the circulation are localized in the liver, phagocytized by Kupffer cells. However, gram-negative septicemia or endotoxemia in humans often leads to acute lung injury (Adult Respiratory Distress Syndrome, or ARDS) and it not understood how pulmonary inflammation develops from systemically introduced pathogens. Sheep and pigs are both used as experimental models for ARDS in humans; possibly, humans develop intravascular macrophage-type cells in the lung after endotoxemia or liver injury, making their pulmonary circulations behave more like those in species with resident intravascular macrophages. To determine if this is possible, workers are attempting to induce a pulmonary intravascular macrophage population in species without the cells by chronic endotoxin infusion (4, 5, 7, 68).

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