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Vol. 83, Issue 5, 1499-1507, 1997
Cardiovascular Research Institute, ABSTRACT Sone, Yasuyuki, Anne Nicolaysen, and Norman C. Staub, Sr.
Effect of particles on sheep lung hemodynamics parallels depletion
and recovery of intravascular macrophages. J. Appl. Physiol. 83(5): 1499-1507, 1997.
liposomes; quantitative histology; Monastral blue particles; phosphonates; pulmonary arterial pressure
INTRODUCTION PULMONARY INTRAVASCULAR MACROPHAGES (hereafter referred
to as macrophages or intravascular macrophages) are a resident
population of mononuclear cells in the lung capillaries of certain
species of mammals, principally those in the orders
Artiodactyla (12) and
Perrisodactyla (11; see Ref. 15 for
review). All of the species having reactive intravascular macrophages
show remarkable pulmonary hemodynamic and acute lung injury responses
to intravenous infusions of endotoxin or gram-negative bacteria
(2-5, 16). Following Winkler's quantitative
histological study (23) of intravascular macrophage development after
birth in pigs, we showed that in newborn lambs the pulmonary
hemodynamic responses to foreign particulate matter (liposomes;
Monastral blue) developed in parallel with the maturation of the
pulmonary intravascular macrophage system. Those results strongly
supported Koch's third postulate about cause and effect (10).
An even better test of cause and effect would be to deplete the
macrophages, show that the pulmonary vascular responses disappeared, and then allow macrophage repopulation to occur and show that the
response returned. No satisfactory depletion method existed until Van
Rooijen and Van Rooijen et al. (18-22) demonstrated that macrophages could be selectively killed in rats and mice by delivering liposomes containing the heavy-metal-chelating agent dichloromethylene diphosphonate [Cl2MDP
(Clodronate)].
Van Rooijen depleted liver intravascular, splenic intravascular,
alveolar, or peritoneal macrophages, depending on the route of
administration and dose of liposomes used. Furthermore, he showed
histologically that the macrophages were destroyed, not merely inhibited. Repopulation of specific organs by newly
recruited blood monocytes that differentiated
into macrophages occurred over 1-2 wk. Thepen and
colleagues (17a) and Berg and associates (1) have confirmed that
>70% of alveolar macrophages can be killed by tracheal instillation
of Cl2MDP liposomes in mice or rats. Hashimoto and co-workers (8) depleted up to 95% of alveolar macrophages in rats by aerosol inhalation of Clodronate
liposomes. Pennanen (12a) fed Clodronate and other diphosphonate
liposomes to cultured mouse macrophage-like cells (RAW 264 cells).
Clodronate was the most effective of the diphosphonates tested in
blocking the cellular secretion of cytokines in response to
Escherichia coli lipopolysaccharide.
To our knowledge, no one has used the liposome depletion method in
large animals.
We have used Van Rooijen's method to deplete the pulmonary
intravascular macrophages in small lambs (~50 times larger than rats). By trial and error, we developed a regime that allows us to kill
the vast majority of these cells. In the present study, we compare the
pulmonary hemodynamic responses to foreign particles among Control,
Depleted, and Recovery lambs (2 wk). Furthermore, by measuring the
retention of Monastral blue (11% copper) in the lungs and by
quantitative electron microscopy, we have confirmed Van Rooijen's (20)
claim that the intravascular macrophages were selectively
destroyed. Within 2 wk, recolonization of the lung capillaries was well
established and the lung hemodynamic response had returned.
METHODS After preliminary tests in which we learned to make and deliver
Clodronate liposomes intravenously, we used thirty-one 3-mo-old lambs
(14.6 ± 3.6 kg) to develop a standardized depletion regime that
selectively killed the intravascular macrophages, particularly those of
the pulmonary microcirculation. Throughout these experiments, we used
sterile techniques insofar as possible, because lambs deprived of their
intravascular macrophages have little defense against sepsis (see
DISCUSSION).
Making Liposomes
After evaporation of the chloroform from the lipid mixture to a dry
film under argon, we added an aqueous solution of disodium dichloromethylene diphosphonate
[Cl2MDP · 2Na · 4H2O
(Clodronate), a gift from Boehringer Mannheim] by dissolving 3.0 g in 10 ml sterile saline (molecular weight 363; 0.82 M) and rotated
the flask under argon until all of the lipid film had spontaneously formed liposomes containing the Clodronate. We made about 20 batches of
liposomes from the 10 ml Clodronate solution, so that each batch
contained no more than 0.15 g (0.4 mmol).
We filtered the milky suspension through a 25-mm-diameter
stainless steel filter holder (Millipore XX3002500) fitted with a mixed
cellulose ester filter (Millipore MF), finishing with two passages
through 0.8-µm-pore-size filters to reduce the liposome size to <1
µm diameter.
We ultracentrifuged the liposomes at 100,000 g and 4°C for 45 min. Because the
liposome layer floats, we removed the underlying solution of excess
Clodronate by using a syringe and a 25-gauge needle,
freezing the excess for reuse in subsequent liposome preparations.
Finally, we resuspended the liposomes in sterile phosphate-buffered
saline (PBS) and stored them in a refrigerator until used (<48 h).
Random cultures for aerobic and anaerobic organisms and the limulus
chromogenic assay for endotoxin were negative. We also made liposomes
that contained PBS but no chelating agent (empty liposomes) to serve as
controls on the effect of the particles alone.
What Are Diphosphonates?
We previously
showed in newborn lambs that the pulmonary hemodynamic responses to
foreign particulate matter (liposomes; Monastral blue) developed in
parallel with the maturation of the pulmonary intravascular macrophage
system. We now report our use of the liposome-encapsulated
heavy-metal-chelating agent dichloromethylene diphosphonate to deplete
the intravascular macrophages of small lambs. Functionally and by
quantitative histology, we depleted the vast majority of the
intravascular macrophages (71% by Monastral blue particle retention,
n = 22; 77% by histology; n = 2). Depletion success increased to
>90% as we optimized the liposome-depletion regime. Recovery of the
lung hemodynamic response began within 3 days. By 2 wk, the functional
responses had fully recovered (n = 8),
and, according to quantitative histology, the macrophage population
(n = 2) had recovered
65%. Macrophage depletion in lambs is relatively
inexpensive and easy to achieve. It is a safe procedure and is followed
by full recovery in ~2 wk, provided that an aseptic technique is used
to prevent bacteremia.
Experimental Protocol
Sheep. In preliminary sterile surgery under 2-3% halothane in oxygen, we placed a Swan-Ganz triple-lumen, flow-directed catheter into the main pulmonary artery via an external jugular vein. We also placed catheters in one carotid artery and into an external jugular vein. After the lamb recovered from anesthesia, we began the depletion process. Groups. There were three groups of lambs: Control, Depleted, and Recovery. The Control group (n = 9) was infused with empty liposomes. The Depleted group (n = 14) was given the Clodronate liposomes over 1 or 2 days. In these groups, we measured microsphere test responses on days 1, 2, and 3 after completing the liposome infusions. The Recovery group (n = 8) was given the Clodronate liposomes and tested for the microsphere responses as in the other groups. We continued to monitor the lambs by doing microsphere tests every few days until the pulmonary arterial pressure response had returned to the baseline level. Liposome infusions. Initially, we were uncertain about how many liposomes we would need. Our 15-kg lambs were ~50 times larger than the rats Van Rooijen used. However, Van Rooijen (personal communication) wrote that he found the liver intravascular macrophages to be far easier to kill than the splenic intravascular macrophages, presumably because of the marked differences between liver and spleen blood flows. Based on the fact that 100% of the liposomes are available for phagocytosis by the intravascular macrophages during the first pass through the pulmonary microcirculation, we reasoned that we ought to be able to use much smaller doses than one might expect based on body weight considerations. After experimenting with various dosage regimes, we achieved maximum depletion by infusing four batches (one batch on each of four occasions over 2 days). We suspended the liposomes in 30 ml sterile saline, which we infused intravenously in 1 h. The number of liposomes infused at each step was about twice the quantity of Clodronate liposomes given by Van Rooijen to rats. Our reasons for using slow multiple infusions include: 1) during the first infusion, the pulmonary intravascular macrophages are stimulated to cause pulmonary arterial hypertension, which may have serious physiological effects (12); 2) the macrophages develop tachyphylaxis during continuous infusions (12); 3) after infusing Monastral blue, we have found at postmortem that some lung units (up to 1-cm diameter) may not be perfused at a particular time (Staub, unpublished data). Macrophage test responses. Our standard test for pulmonary intravascular macrophage reactivity is to infuse over 1 min 400 µl of a 1% suspension of 1-µm-diameter polystyrene microspheres (Duke Scientific, Burlingame, CA) in 10 ml of saline. In normal sheep, this dose of test particles gives a transient rise in pulmonary arterial pressure, beginning ~30 s after commencement of the infusion, peaking within 2 min, and recovering to baseline within 5 min. This stereotypical response is dependent on the presence of reactive macrophages in the pulmonary capillaries and is due to rapid synthesis and release of thromboxane by the intravascular macrophages (10, 12). Monastral blue retention. Monastral blue pigment particles are well suited to quantifying the pulmonary intravascular macrophages. In normal mammals, particles infused intravenously are specifically removed from the circulation by intravascular macrophages, presumably in proportion to organ blood flow. By gross examination, light microscopy, or by electron microscopy, the Monastral blue particles are easy to see (bright blue and electron dense). On the day after we had completed all liposome infusions, we infused 5 mg/kg of Monastral blue (Sigma Chemical, St. Louis, MO)1 diluted to 30 ml with saline and infused over 30 min. When Monastral blue infusions are used consistently in this manner, the lungs of Control sheep are bright blue (after the residual blood has been washed out), while the lungs of well-depleted sheep are only faintly gray blue. We saved a sample of the injectate to determine its copper concentration and weighed the infusion syringe before and after use so that we could calculate the amount of Monastral blue (copper) infused. In several lambs, we took systemic arterial samples at 1, 2, 4, 8, and 16 min after the Monastral blue infusion was completed to measure the rate of clearance from blood (an additional independent measure of phagocytosis by intravascular macrophages). In normal sheep, phagocytosis by the intravascular macrophages is so efficient that the 1-min sample is completely free of Monastral blue. Quantitative histology. Lambs were killed in the following manner. We anesthetized and ventilated each lamb, placing it in the dorsally recumbent (supine) position. Through an external jugular venous catheter, we infused (during 2-3 min) 1 liter of PBS while we exsanguinated the animal via severed femoral arteries. In two animals of each group, we opened the chest, perfused the lungs with another liter of PBS, fixed the lungs by perfusion with 2.5% glutaraldehyde-1.5% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4), and made five stratified random blocks from the right and the left large caudal lobes (equivalent to lower lobes in humans). The lung tissue was further processed for conventional transmission electron microscopy. After preliminary sampling and discussion, we decided to count the number of capillaries containing macrophages. From the 36 embedded tissue blocks of each lamb, we chose six random blocks (three each from the right and the left lung) and examined one ultrathin section (60 nm thick, poststained with uranyl and lead) that had been put on a 200-mesh grid. We followed consecutive grid meshes until we had examined 50 fully visible capillaries along the alveolar walls and recorded the number that contained macrophages. Thus we examined 300 lung capillaries from each block. For signs of cellular toxicity, we also examined the endothelium, the adjacent alveolar epithelium, and the few residual intravascular leukocytes that had not been washed out by our fixation process. Quantification of Monastral blue. We weighed each lung, added an equal quantity of water, then homogenized the combination in a large commercial Waring blender. We digested duplicate weighed samples of each lung homogenate in 10 g concentrated nitric acid to destroy the Monastral blue, release the copper, and dissolve the tissue. The injectate samples were similarly treated, except that they were quantitatively diluted 100-fold. The digestates were stored in a freezer until we had sufficient numbers to analyze. We did the analyses by using an atomic absorption spectrometer (model 151, Instrumentation Labs, Lexington, MA) that was linearly calibrated over the expected range of our samples.Statistics
The group data are summarized as the mean ± 2 sample SD. In some places, we compare macrophage depletion in relative units (%). In the quantitative histology series, we maintained a running mean and SE for each lung. We used a one-way analysis of variance among groups, then an unpaired t-test between groups, with Bonferroni's correction for repeated measures. We did this for completeness, because the results are so obvious that no statistical analysis was necessary.RESULTS
Macrophage Depletion
We tested various liposome quantities and infusion regimes. We began with one batch (16.4 µmol phosphatidyl serine or 0.4 mmol Clodronate; n = 2; see Making Liposomes for details), then two batches (n = 7), and finally infused four batches over 2 days (n = 13). Totaldepletions included 22 lambs. The maximum quantity of liposomes given was eight times the dose used by Van Rooijen (20) to deplete the liver intravascular macrophages (Kuppfer cells) in rats. In the Control lambs (n = 6), we infused the empty liposomes in two or four doses; three other Control lambs received no liposomes, only an equivalent volume of saline.
Time course. Figure 1 shows representative examples of the pulmonary arterial pressure response to our test microspheres in three lambs. The response of the Control lamb (Fig. 1A) was not significantly altered by giving the empty liposomes. The response of the Depleted lamb (Fig. 1B) shows that, after the first day's treatment, the response to our test dose of plastic microspheres was decreased ~50% and was completely eliminated after the second day's treatment. On the fourth day, the response was still absent. The response of the Recovery lamb (Fig. 1C) shows that the test response reappeared within 3-4 days and returned to the control level within 10 days.
Blood clearance of Monastral blue. We used this as a nondestructive and independent way to assess inhibition of one of the prime functions of macrophages. In every Control lamb, we took an arterial blood sample 1 min after completing the pigment infusion. None showed any color in the plasma, and, by quantitative analysis, there was no copper above background levels (see large gray symbol with SD bars in Fig. 2). The figure also shows semilog plots of plasma copper concentration for four Depleted and two Control lambs in which we followed clearance from blood plasma for 16 min. In the Depleted animals, plasma clearance half-time was ~30 min, compared with <1 min in the Control lambs. We did not do this test in the lambs after Recovery because we did not want to confound the lung copper data from the initial Monastral blue infusion after macrophage depletion.
Quantification of Intravascular Macrophages by Electron Microscopy
Although the fixed lung samples were coded for single-blind analysis, the bright blue Control blocks were obviously different from the others. However, the Depleted blocks and the Recovery blocks had a similar gross appearance. The perfused-fixed lung is nearly ideal for intravascular macrophage quantification because the capillaries are distended and all but a few erythrocytes and circulating or sequestered leukocytes were washed out.Sheep pulmonary intravascular macrophages are ~25 µm in extent, with at least six branched pseudopodia extending 5-10 µm into multiple capillary segments and with focal adhesion plaques between the cell and the underlying endothelium in the nuclear region of the cell (13). In an ultrathin section (60 nm) of lung tissue, most capillaries show one or more pieces of macrophages, either cut through the nuclear region or through pseudopodia. It is irrelevant to our study whether macrophage pieces in the same or the adjacent capillaries were from the same or different cells. We studied one ultrathin section from six different regions of the lung in each animal. Figure 3 shows representative examples of low-power electron micrographs to give an overall impression of these lungs. In the Control lung (Fig. 3A), the majority of capillaries contain pieces of macrophages. The Depleted lung (Fig. 3C) contains no identifiable pieces, although there is one possible disintegrating macrophage in one capillary (arrow). The Recovery lung (Fig. 3D) contains many pieces of macrophages but less than the Control lungs. In the Control lungs, 25% of macrophage pieces contained Monastral blue phagosomes (Fig. 3B), whereas in the Recovery lungs no more than 2.5% of macrophage pieces contained Monastral blue. The timing of our Monastral blue infusion is important. We infused the Monastral blue the day after we completed depletion and before recolonization occurred. The data are summarized in Table 1, in which we have listed the percentage of 300 capillaries counted that contained one or more identifiable pieces of macrophages. Figure 4 shows the cumulative means ± SE of macrophage counts in the three sample groups. The final means ± SD are independent of the order in which the sections were counted. The data are so markedly different between Control and Depleted lungs that the results were statistically significant after only 50 sections were counted. However, for completeness and to sample several areas of each lung, we continued to count 300 capillaries.
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In relative terms, the number of intravascular macrophages was decreased by 67-84% (mean 76%) after the Clodronate liposomes. The lamb showing the lesser depletion was infused early in our experiments; it received only two batches of liposomes. The lamb showing the greater depletion was infused late in our experiments. It had received four batches of liposomes. For both Recovery lambs, the quantitative cytology showed 65% as many intravascular macrophage pieces in capillaries compared with the mean of the Control lambs.
Quantification of Intravascular Macrophages by Lung Monastral Blue Retention
Table 2 summarizes the lung Monastral blue content, based on the copper content measured by atomic absorption analysis. Because there is a small amount of copper, averaging 1.03 µg/g, in normal sheep lungs that have not received any Monastral blue (unpublished data), we have subtracted this quantity from each lung. The correction has essentially no effect (~2%) in the Control lungs because of the enormous amount of copper from Monastral blue in the lungs. The correction is greater in the Depleted and Recovery groups because a constant quantity of copper is being subtracted from a diminishing total quantity.
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Overall macrophage depletion averaged 72% compared with normalized Controls in which the average was 100% (P < 0.01). However, we knew that early on we had used fewer batches of liposomes. Therefore, we compared the lungs of those lambs that had only received one to three batches of Clodronate liposomes (n = 9) with the lungs of those that had received four batches (n = 13). The former showed 62.6 ± 18.8% depletion compared with the latter, which showed 88.8 ± 4.6% depletion (P < 0.01). The differences between Control and all Depleted (or Recovery) lambs were highly significant. There was no data overlap between the groups. The Monastral blue content of the two Depleted lungs averaged 11.2% (22.3 and 5.8% of injected dose, respectively), whereas in the same lungs quantitative histology averaged 23.0% (27.2 and 18.4% of the mean of the two Control sheep lungs). Unfortunately, no internal control is possible on the histology. Nevertheless, both methods show extensive depletion.
The Monastral blue content of the two Recovery lungs averaged 20.6% (26.4 and 11.9% of the injected dose, respectively), whereas quantitative histology in the same lungs averaged 64% (64.4 and 64.2% of the injected dose in each sheep), respectively. Thus most of the Recovery macrophages did not contain any Monastral blue phagosomes, which one would expect if the macrophages had differentiated from recolonizing monocytes. We also tested whether there might be some loss of lung Monastral blue content over the 2-wk Recovery period. We compared six Depletion with seven Recovery lambs, all treated with the four-batch depletion protocol. The lung Monastral blue contents averaged 14.2 ± 3.7 and 9.3 ± 4.0% of the infused dose, respectively (P < 0.1, that is, no statistical difference).
DISCUSSION
The functional, chemical, and electron-microscopic data presented in RESULTS are so disparate among the groups that no discussion is required; the data speak for themselves. However, several ancillary points about macrophage function can be made.
Protection Against Bacteremia
Lambs depleted of macrophages are unprotected against septicemia. Among our seven pilot lambs (data are not included in this report), we found by blood culture that two had sepsis. Because they did not respond to any treatment over 24 h, we had to kill them by anesthetic overdose. After adopting strict aseptic measures, we had no further loss due to sepsis. We believe protection against infection, especially bacteremia, is of major importance when using macrophage depletion.Cells Normally Phagocytizing Particles in Blood
In addition to macrophages, we examined our perfused-fixed lung sections for cells that may have phagocytized test microspheres, liposomes, or Monastral blue. Although our fixation procedure washed out nearly all of the leukocytes, we did find a few neutrophils; none contained any particles. In an earlier study in which we washed out the lungs of goats that had received Monastral blue intravenously (16), we collected and made cytospins of samples of the myriad leukocytes that we obtained. Among all our samples, we only found two neutrophils that had ingested a few tiny Monastral blue particles.These data are consistent with reports that circulating leukocytes need to be primed by trace quantities of endotoxin before they become adherent to endothelium or phagocytically active (7, 9). We infer that the sheep we studied did not have circulating endotoxin and that their circulating or sequestered leukocytes were not phagocytic.
We searched diligently but never found any evidence of phagocytosis of Monastral blue, liposomes, or microspheres by lung endothelial cells.
Lung Cells That May Have Been Injured by Clodronate
Except for the intravascular macrophages, we never found any morphological evidence of endothelial, alveolar epithelial, or intravascular cell injury. This is consistent with the fact that free Clodronate does not harm cells and that only the macrophages normally phagocytize intravascular particles.Blood Clearance of Particles
We infused our standard dose of 5 mg/kg Monastral blue slowly (30 min) because the particles stimulate the pulmonary intravascular macrophages to produce and secrete thromboxane in large amounts that are sufficient to cause severe pulmonary hypertension (12). At the end of the infusion, an arterial blood sample never contained any detectable pigment particles. However, after macrophage depletion by our Clodronate liposomes, arterial clearance was markedly delayed. Furthermore, after depletion, there was no hemodynamic response to any particulate infusions.Why does any clearance occur at all, if there is no mechanism other than phagocytosis for particle clearance from plasma? First, we did not kill all of the intravascular macrophages. Even in our best depletion experiment, ~5% of pulmonary intravascular macrophages survived; the average was 28% survival. Probably even more of the liver macrophages survived, although we did not specifically examine them. Furthermore, Van Rooijen (19) stated that splenic macrophages are very resistant to depletion by Clodronate liposomes, presumably because splenic blood flow is so low compared with lung or liver that few liposomes reach the spleen. Thus surviving liver, splenic, or bone marrow macrophages probably account for the nonzero clearance of Monastral blue.
Were the Intravascular Macrophages Really Killed?
Our chosen method of counting the number of capillaries containing macrophages is a relative measure; it does not give the absolute volume fraction of capillaries containing macrophages (23). One could argue that, by contracting into a sphere, fewer capillaries would retain pieces of each macrophage. This possibility is unlikely to explain the drastic reduction in the macrophage counts that we recorded. The pulmonary intravascular macrophages of sheep may be 25 µm in extent, because they send pseudopods into many neighboring capillary segments (13). However, if the cell retracted into a sphere, its volume would be enormous. Thus, while changing shape may somewhat reduce the fraction of capillaries containing pieces of macrophage, it is not likely that a shape change alone could reduce the fraction by as much as we found.In the Depleted lungs, ~25% of the macrophage pieces that we counted appeared to be nonviable by morphological criteria (surface blebs, ruptured membranes, and diffuse cytoplasmic structures). In addition, we occasionally found remnants of macrophages (see arrow in Fig. 3C). We scarcely ever found such disintegrating or remnant cells in the Control or Recovery lungs. These histological findings do not fit the retraction explanation.
Furthermore, changing macrophage shape would not reduce the Monastral blue content of the lung; this reduction was dramatic after depletion (Table 2). The presence of some dead or dying macrophages would also allow us to explain why the histological data for the Depleted lungs showed more macrophages than did the Monastral blue retention data. We do not have to invoke the lack of internal controls on the histology to explain the difference. However one interprets the data, we cannot escape the main conclusion that by all measures of lung intravascular macrophage function, we markedly reduced by the Clodronate-containing liposomes.
Any macrophages containing Monastral blue that we found in the Recovery lungs must have been present at the time of the Monastral blue infusion. Because the Recovery lungs contained few Monastral blue phagosomes, the logical explanation is that circulating monocytes, which do not normally phagocytize Monastral blue, had recolonized the lung and differentiated into macrophages during 2 wk (10). In a review of lung colonization in piglets, Winkler (23) stated that at birth the lungs contained only monocytes, by morphological criteria, but by 7 days there were many cells with macrophage characteristics. We examined the capillaries in the Recovery lungs and found that ~6% of the macrophages could be classified as monocytes; this supports the recolonization concept.
Regeneration of functionally damaged but viable intravascular macrophages may have occurred; cell division of the surviving macrophages is another possibility. In both instances, we would have expected the cytoplasm to contain the originally deposited Monastral blue and, during division, any ingested dye particles ought to have distributed more or less evenly between the daughter cells.
Furthermore, macrophages are considered fully differentiated cells and usually do not divide actively. The response to foreign particles was completely restored at a time when the quantitative analysis of macrophages in lung capillaries had not entirely returned to the Control condition. We interpret that to mean that the new macrophages, although functionally active, had not reached their maximum size.
The elimination of the responses when the macrophages were depleted and the restoration of the responses when the macrophages recovered fulfills Koch's third postulate for cause and effect. These results go hand-in-hand with our earlier developmental data about the hemodynamic responses to foreign particles and the maturation of the pulmonary intravascular macrophage population in newborn lambs (10). We conclude that the pulmonary hemodynamic response to foreign particles is dependent on the presence of reactive pulmonary intravascular macrophages.
ACKNOWLEDGEMENTS
We thank Boehringer Mannheim for its generous gift of Clodronate.
FOOTNOTES
Address for reprint requests: N. C. Staub, Sr., PO Box 965, Stinson Beach, CA 94970.
Received 24 February 1997; accepted in final form 13 June 1997.
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