European Journal of Clinical Investigation ( 1 986) 16, 500-508

Evaluation of low dose anaphylatoxic peptides in the pathogenesis of the adult respiratory distress syndrome (ARDS). Monitoring of early C5a effects in a guinea-pig in vivo model after i.v. application*

T. HOFFMANN,* E. C. BOTTGER,* H. P. BAUM,? R. DENNEBAUM,$ U. HADDING* & D. BITTER- SUERMA",* Institute of Medical Microbiology,* Institute of Pathology,? and Department of Clinical Chemistry and Laboratory Medicine,$ University of Mainz, 6500 Mainz, FRG

Received 17 March 1986 and in revised form 2 July 1986

Abstract. A guinea-pig in vivo model is presented that allows the infusion of purified C5a via a central vein catheter and the monitoring of its effects on granulo- cytes and platelets, the most important cells in the pathogenesis of several lung disorders, e.g. shock lung. After the infusion of C5a, which was adjusted to a quantity that caused slight and transient alterations of lung physiology, granulocytes disappeared from circu- lation within 1 min. Simultaneously the granulocyte content of the lung increased about three-fold as judged by histological evaluations. Morphologic des- tructions were not observed. After the drop a rebound of circulating Polymorpho-nuclear leucocytes (PMN) occurred, which was significantly higher than control values and the appearance of banded cells indicated a mobilization from bone marrow stores. Studies with 5 1 -chromium labelled PMNs revealed that most, but not all, of the granulocytes returned to circulation after transient sequestration. The number of platelets also decreased after C5a infusion, but the rebound was delayed compared with the PMNs and did not exceed control values. The changes in circulating cells, lung histology, and lung physiology are comparable to those occurring during the onset of shock lung and thus strengthen the supposed importance of C5a concerning the pathogenesis of that syndrome.

Key words. C5a, granulocytes platelets, respiratory dysfunction, guinea-pig.

Introduction

Due to the improvement of life-supporting systems today the acute phase of different life-threatening disorders can be well managed. On the other hand

severe complications, which in former times rarely developed because of early death of the patients, gained increasing importance in recent years. One of these complications represents the adult respiratory distress syndrome (ARDS) with an overall mortality of approximately 50% [I].

In the pathogenesis of acute lung dysfunction like this syndrome, the anaphylatoxic peptide C5a, minor split product of the complement component C5, and its first degradation product C5a-des Arg, the C5a- peptide without the C-terminal arginine, are consi- dered to play an essential role. According to current concepts such different clinical events as sepsis due to gram-negative microorganisms, polytrauma, pancrea- titis, and others lead to massive activation of the complement system often far away from the lung. Generated C5a acts on neutrophils and causes them to stick in the capillary bed of the lung. The activated neutrophils then injure the endothelium by generation of toxic oxygen radicals, arachidonic acid metabolites, and liberation of proteases that destroy structural proteins. This scenario leads to an increased capillary permeability, the starting point of histologically well investigated derangements possibly leading to the typical shock lung [for review see 1-51.

The contribution of complement, particularly C5a, to the outlined pathophysiological sequelae has been investigated by numerous authors employing different animal models. However, with one exception [6] most approaches which want to demonstrate the influence of C5a do not imitate the supposed way in which C5a acts in the genesis of ARDS: (i) some investigators injected cobra venom factor (CVF) into animals thus activating total complement rapidly and without con- trol [7, 81; (ii) others employed purified anaphylatoxic DeDtides but instilled them in hiah doses intratra- chkally, a rather non-physiologic method (9, 10); (iii) Huey et al. [ I I] injected purified C3a in an extremely high dose intravenously together with an inhibitor of the Serum carboxypeptidase, protecting C3a from inactivation, which led to acute bronchospasm or

Correspondence: D. Bitter-Suermann, Institute of Medical Microbiology, Augustusplatz, D-6500 Mainz, FRG.

* Parts of this work were presented at the Inflammatory Media- tors Symposium, 4 August 1984, a t the Royal College of Surgeons, London, U.K.

500

THE ROLE OF C5a IN EARLY STAGES O F ARDS 501

death within 2 min. These effects are not comparable with clinical signs during the onset of ARDS.

For these reasons we developed an in uivo guinea-pig model, which enabled us to infuse purified C5a via a central vein catheter in doses which did not cause acute, severe clinical symptoms and to remove blood samples at different times up to three days to study in a time course (monitoring) the behaviour of granulo- cytes and platelets, the main effector cells at the onset of lung failure. Histologic examinations of the lung were done in parallel to evaluate the corresponding morphological alterations.

Materials and Methods

Animals Guinea-pigs of inbred strain 2 NIH and 13 NIH

were held in our own colony. All animals had 400-600 g body weight when used for experiment. Results were totally independent of either strains, two or thirteen guinea-pigs, and males or females were used.

Guinea-pig C5a and CSa-des Arg

C5a was prepared according to published methods [12]. The resulting material was tested for purity on IOY” SDS-PAGE. After silver staining (Bio-Rad, Richmond, Calif.) it appeared as a single band with a molecular weight of approximately 14 000.

The des Arg variant of C5a was prepared by incubating the pure anaphylatoxin preparation with carboxypeptidase B (Boehringer-Mannheim, Mann- heim, FRG), the latter at a final concentration of 200 pg ml- I in a buffer containing 0.002 m Ca+ + and 0.001 m Mg++ (as chloride) for 15 min at 37°C.

Central vein catheterizalion All surgical procedures were performed under keta-

minhydrochloride-anesthesia (100 mg kg- ’ body- weight f0.05 mg kgg atropine both injected together intramuscularly). Animals were restrained by softly tying each limb and the head to a board. The ventral neck region was cleaned, and approximately I ml of 1 % lignocaine was injected subcutaneously beside the throat region for preference on the right. There, close to the midline, a cephalo-caudal incision was made and the tissues were spread by blunt dissection. The external jugular vein was prepared, elevated, and a section of about 1.5 cm was cleaned. A silk ligature was tied around the cephalad end of the vein, and a loose overhand knot placed at the caudal end. While the vein was pinched off by a pair of tweezers at the caudal end to prevent air embolism, a small incision was made. A catheter made of polyethylene with an outer diameter of 1.5 mm and an inner diameter of 1.0 mm, length 38 cm (Braun-Melsungen, Melsungen, FRG) was inserted and advanced toward the heart for a distance

of about 5 cm. Throughout this procedure the catheter was gently rinsed with isotonic saline to prevent any coagulation. After several knots had been tightened fixing the catheter in the vein together with the surrounding connecting tissue, the catheter was guided subcutaneously to the dorsal neck and from there outside the animal through a small incision in the skin. The end was attached by an intermediate three-way stopcock to a disposable syringe filled with isotonic saline. Wounds were closed by several Miralene- ligatures (Braun-Melsungen, Melsungen, FRG). Ani- mals were then put into a small box (10 x 30 cm) and allowed to recuperate overnight. The next day, when they were in good condition as indicated by eating and drinking, experiments were started. Throughout all the time a constant flow of saline (0.6 ml h-’) achieved by an infusion pump (Braun-Melsungen, Melsungen, FRG) was maintained to keep the tube open.

Application and blood collecting procedures. All injections and the removal of blood were made via the three-way stopcock. C5a was dissolved in 1 ml sterile, pyrogen-free saline and injected over a time-period of 30 s. The endpoint of this injection was taken as time zero. For control experiments I ml saline was injected without C5a. After each removal of blood the catheter was rinsed with 2 ml saline. Bloodcell counts including a differential leucocyte count were done by a multi- channel auto-analyzer (Technicon H 6000, Technicon GmbH, Bad Vilbel, FRG). Because this apparatus cannot distinguish between banded and segmented granulocytes, 200 cell differential counts were per- formed by standard techniques where indicated in the text.

Correction factor. Since there occurred an artificial dilution of circulating blood by repeated injections of saline during the course of the experiment, a correction factor was introduced: Ecl + Ec2/2 x En, (Ec,, Ec2 = er- ythrocytecounts of two control samples, drawn 10 min and 1 min before starting the experiment; En = eryth- rocyte count of sample n) . All cell counts of sample n, except erythrocytes, were multiplied with this factor.

Preparation and labelling of granulocytes. Granulo- cytes were prepared from the heparinized blood of a guinea-pig from the same inbred strain as the animal in the experiment. This blood was drawn by cardiac puncture and PMNs were separated using Ficoll- Hypaque (Pharmacia, Uppsala, Sweden) density gra- dient centrifugation, as described by B ~ j u m , [13]. Cells were suspended in PBS and adjusted to a density of 2 x lo7 cells ml-I. Normally about 1 ml was obtained with more than 98% PMNs. They were labelled by incubation with 100 pl Chromium-chromat ( 5 mCi/ m1-l) (Amersham, Braunschweig, FRG) for 45 min at 37”C, followed by two washing steps with PBS. After these procedures we normally obtained a total cell amount of 1-1.5 x lo7 granulocytes, more than 97% of which were viable as judged by trypan-blue exclusion.

502 T. HOFFMANN et al.

Radioactivity counting

Radioactivity of whole blood samples, (200 pl blood per vial) reflecting the content of 5 l-chromium- labelled granulocytes, was measured in a Packard Spectrometer Mod. 5760.

Measurements of lung physiology Lung function (breathing rate, dynamic compliance,

resistance) was measured in the laboratory of Dr A. Fugner, Boehringer, Ingelheim, as previously de- scribed in detail [14]. After the animal had been provided with a tracheal cannula and a water-filled esophageal catheter, it was placed in a supine position into a body plethysmograph. After this procedure half an hour was left for adaptation of the spontaneously breathing animal, while normal lung function was checked intermittently. Starting 5 min prior to the i.v. injection of C5a, measurements were performed conti- nuously for about 30 min.

Fixation of the lung Under deep ketanest narcosis, the abdomen was

opened by a ventral cranio-caudal incision, the intes- tine gently moved aside and the distal aorta exposed. At a certain time interval after the injection of C5a, 4% phosphate buffered formalin solution (pH 7.3) was infused via the central vein catheter (25 mmHg, 30 min, about 300 ml total volume). Simultaneously the abdo- minal aorta was slit. After fixation the lung was excised without signs of collapse. It was postfixed by immer- sion in buffered formalin solution overnight and processed by routine histological techniques. Paraffin sections (4 pm) were stained with hematoxylin and eosin.

Morphometric studies From each animal five pulmonary histological sec-

tions comprising central and peripheral parts of the lung were evaluated. In a total of 150 high power fields the number of PMNs and capillary endothelial cells were determined. Calculating the ratio of granulocytes to endothelial cells compensated for different degrees of expansion of the pulmonary tissue. Preliminary tests had revealed that this ratio reached its final value (k 1 %-2%) after counting 40-60 fields.

Statistics Because the observed cell counts were log normal

distributed, geometrical mean values and the 95% confidence interval or the 95% tolerance interval were computed. The hypotheses were tested by non-para- metric methods like the Mann-Whitney U-test.

Results

Control experiments

The performance of the experimental procedure with five animals in which 1 ml saline was injected instead of C5a yielded the results outlined in Fig. 1 and Table 1. At each indicated time 1 ml of blood was removed and the catheter was subsequently rinsed with 2 ml saline as was done in other experiments with C5a application. As can be seen, the granulocyte, platelet and erythrocyte counts steadily declined. The last reached about 65% of the starting value after 5.5 hours indicating a dilution effect by the total loss of 13 ml blood and the administration of about 26 ml saline. Therefore, PMN and platelet counts were multiplied with a correction factor as described in Materials and Methods. The corrected PMN counts showed no significant alterations throughout the experiments, whereas the platelet counts revealed a slight increase.

1 7 o i

i

6 0 701 5 0 1 k--u-

-100 815 30 60 90 150 240 330 t T ime (rnin)

I rnl sal ine

Figure 1. Effects of repeated removals of blood on circulating cells in control animals. At each indicated time, 1 ml of blood was removed and the catheter subsequently rinsed with about 2 ml saline. The arrow indicates injection of 1 ml saline instead of AT-peptide. Values are means from 5 animals & 1 SD. Broken line: loo%, baseline value. A, measured value; 0, value corrected for dilution. From the measured erythrocyte values, the correcting factor for each time was calculated (see material and methods). The measured (= uncor- rected) values of neutrophils and platelets were then multiplied by the correcting factor to produce the corrected values. Correcting the values did not alter the standard deviations (SD).

THE ROLE O F C5a IN EARLY STAGES OF ARDS 503

Table 1. Effects of saline, C5a and C5a-des Arg on circulating celk in the peripheral blood of guinea-pigs

Animal Stimulus no.

Saline 1 (control) 2

3 4 5

C5a 6 7 8 9

10 11 12 13 14

CSa-des Arg 15 16 17

~~ ~ ~~

Number of neutrophils Number of platelets ~ ~~ - -. . .-..

Before Minimum Maximum Before Minimum Maximum stimulus (1 rnin after (between 1 hand 4 h stimulus (Between 1 min and 4 min (Between I hand 5.5 h I = O stimulus) after stimulus) t = O after stimulus) after stimulus)

7800 7700 8600 245 000 238 000 257 000 4350 4200 3550 270 000 250 000 315000 6500 7250 10 450 460 000 478 000 688 000 5300 5190 5300 222 000 251 000 277 000 3700 4000 4500 257 000 286 000 318 000

4000 100 10 300 185 000 63 000 268 000 6400 800 1 1 300 395 000 325 000 450 000 2830 120 4200 150000 67000 I78 000 5200 150 10 000 195000 83000 270 000 6500 800 8800 300 000 I60 000 340 000

326 000 6240 250 10 200 234 000 107 000 4650 430 10 600 300 000 158 000 322 000

12 200 800 10 600 280 000 I85 000 265 000 5500 1890 7080 n.t. n.t. n.t.

7350 580 17 580 176 000 185 000 306 000 4850 450 8350 186 000 195 000 240 000 7640 280 20 300 267 000 275 000 263 000

n.t.: Not tested.

CSa-effects on lung physiology The dose of C5a administered intravenously was

adjusted to a quantity that, on the one hand, exerted a major effect on circulating granulocytes (see below), but on the other hand caused no dramatic alterations to the animal's respiratory status. This dose was found to be 1.5 pg kg-' body-weight, in molar terms 2.7 x mol-' in the plasma of the test animal. It corresponds to a quantity present in about 0.3 ml of fully activated plasma or to 1% of the maximal achievable C5a in plasma. To assess the influence of this small quantity of C5a on sensitive parameters of lung physiology we measured the effects on dynamic

compliance, resistance, and breathing-rate. In an experimental group of four animals, the C5a injection was immediately followed by a decrease of dynamic compliance (63%, 48%, 67%, 59%, respectively), an increase of resistance (141%, 155%, 138%, 14776, respectively) and of the breathing rate (1 62%, 182%, 145%, 172%). Maximal alterations in percent of the starting values are given in brackets for each animal. They occurred within the first 2 min after stimulus application returning to normal values shortly there- after. In Fig. 2 the time course of lung function after C5a-injection is depicted for a representative animal (animal 1 , first value of all 3 applied measurements).

Breathing rate 160 - Resistance 150 5

.u 140 130 120

c 100

--- Dyn. compliance I

g 110

P 90 5 80

0, 70 r

5 60 50

0 1 2 3 4 5 6 7 8 I-,

C5a, Time (rnin) injection

Figure 2. Measurements of pulmonary resistance, dynamic compliance, and breathing rate. Mean of the three values preceding the C5a injection (1.5 pg kg-') were set to 100%, all others related to this baseline. Data for a representative animal are given.

504 T. HOFFMANN et al.

Efects of a bolus injection of C5a on circulating granulocytes and platelets

The injection of purified C5a (1.5 pg kg-') caused a rapid decrease of granulocyte and platelet counts (Fig. 3A and Table 1). Within 1 rnin nearly all PMNs disappeared from circulation. After two minutes the PMN count increased reaching the starting value between 8 and 30 rnin and moved up to approximately double the amount after I-? h. This rapid increase was mainly based on stabs (600-900%0 over baseline) indicating a release from the bone marrow. After 6-7 h the granulocytes declined to normal values. The platelets behaved similarly, although only about 50% disappeared and the rebound exceeded control values only slightly (20-30%), not more than in control animals which received saline instead of C5a. The same dose of CSa-des Arg exhibited the same effects on circulating granulocytes as did the C5a. However, platelets remained unaffected (Fig. 3B and Table I) . The data shown are representative of 9 (for C5a) and respectively 3 (for C5a-des Arg) animals. Whereas the extent of neutropenia was almost identical (about 90% reduction), the following neutrophilia varied consider-

Y L

5 10000 a, z

8 000

6 000

L 000

2 000

al al - +.

300

200

100

.... .... ..........,............ , ......................... -10 0 8 15 30 60 150 2LO 330 420

0

t Time (min) C5ades Arg I 1.5pgIkg 1

Figure 3. Reaction of circulating PMNs, stabs and platelets after injection of C5a and C5a-des Arg. Fig. 3A: C5a; Fig 38: C5a-des Arg. dose of AT-peptide: 1.5 pg kg-' in each case. 0, segmented PMNs; A, banded PMNs; 0 , platelets. At time - 10 and 0 blood- samples, serving as controls, were removed. All values are corrected for dilution. Data of representative animals for each stimulus are given.

ably from animal to animal (120-300% of baseline values). Basic data of the individual animals treated with saline, C5a and C5a-des Arg are given in Table 1, where cell-counts before, shortly after (minimum), and 1-5; h (maximum) after stimulus are depicted.

Minima of neutrophils in the C5a- and C5a-des Arg group as well as the minima of platelets in the C5a group are significantly lower than in the saline group ( P < 0.01). Maxima of neutrophils in the C5a group are significantly higher (P < 0.01) than in the saline group. Maxima of neutrophils in the C5a-des Arg group seem not to be significantly higher (P=O.O52) than in the saline group, solely due to the small number of animals (only 3) taken for statistical analysis. But looking at the basic values (Table 1) they show explicitly strong maxima compared to the C5a and saline groups.

Studies with 51 -chromium-labelled granulocytes After injection of labelled granulocytes via the

central vein catheter, the kinetics of radioactivity in the blood showed an initial phase of equilibration for about 1 h, before it vanished in a second phase according to an exponential fashion indicating a random movement of granulocytes out of circulation. Curves plotted on semi-log graphs showed a linear decrease in this phase and clearance half-times could be determined in five animals as 6.0, 7.0, 7.5, 8.4, and 9.2 h (data not shown).

In experiments in which C5a was applied as stimulus (Fig. 4), labelled granulocytes were allowed to equili- brate in circulation for about 1 h and then the phase of exponential decrease was monitored (-60 to 0). After this period 1.5 pg kg-' C5a were injected which caused, in each of four animals, an immediate drop of activity in the blood. This effect was reversible. Already 4 rnin after stimulus application about three-quarters of the vanished activity had returned, one quarter was miss- ing for the next 45 min. One hour after stimulus no further difference between measured and extrapolated curve was detectable.

Histologic examinations and morphometric studies

Histologic examination of lung sections from five animals fixed at 1 rnin after C5a application revealed a clearly higher content of granulocytes than did control animals which had only received saline. These granulo- cytes were not distributed homogeneously, but they had accumulated in distinct regions. To quantify the granulocyte content morphometric studies were per- formed. As can be seen from Fig. 5, there was a three- fold increase in the number of PMNs 1 rnin and 30 min after C5a injection compared to control lungs (40 and 41, respectively vs. 14 PMNs per 100 endothelial cells). Thereafter, the granulocyte content gradually dec- lined. Ninety minutes after C5a 27 PMNs/100 endo- thelial cells were counted and after 3 h only 22 indicating a slow return to control values. The alveolo- capillary walls remained delicate. There were no signs

THE ROLE OF C5a IN EARLY STAGES OF ARDS 505

2400 - 2000 -

1600 -

0 1200-

- 800

Loo -

-60-40-20 0 630 60 90 120 150 180 2LO 300 360 420 LBO t t Time (min)

51Cr -labelled 1.5pglkg C5a PMNs

Figure 4. Effect of C5a on radio-labelled PMNs in the circulation. A total of 1.5 x lo5 PMNs, labelled with 5 1-chromium in vitro, was injected via thecentral vein catheter. After about 120 minutes, C5a was injected. Radioactivity was counted from whole blood (200 jd blood per vial). The dotted line was extrapolated from four values before C5a-injection, according to the exponential fashion that had been observed in three separate control experiments. Its decrease represents the loss of radioactivity due to random movement of granulocytes from the circulation. Data of a representative animal are given.

PM Ns 100 endothelial cells

Control l m i n 30min 60min 9Omin l8Omin 2Lh

n.3 n . 5 n.3 n = 3 n.5 n = L n = 5

Figure 5. Number of PMNs per 100 endothelial cells in the lung at different times after C5a-injection. Abscissa: time after C5a- injection, when the animal was sacrificed. For details see Material and Methods.

of a general smooth muscle contraction of arterioles or bronchioles.

Discussion Evidence has been accumulated that complement peptides play a major role in the pathogenesis of several forms of lung dysfunction, predominantly of ARDS. According to investigations of Craddock et al. [ 151, who studied patients during hemodialysis, which can sometimes provoke acute shock as well as chronic fibrotic lung alterations resembling the fibrosis in the terminal stage of shock lung, the main mediator was judged to be C5a. Confirming these results, Ham- merschmidt et al. found in sixty-one patients at risk of ARDS (major non-thoracic trauma, fungemia, gram- negative bacteremia, severe hypotension) a highly significant correlation between the presence of ele- vated C5a levels in the plasma, measured as aggrega-

tory activity, and the development of ARDS [16]. These investigations prompted quite a lot of work with animal models in which whole plasma complement was activated either in vitro by zymosan, inulin or cellophane and reinjected in the animal or in uiuo by infusion of cobra venom factor [8, 1 1, 17-19]. Stimler et al. instilled purified C5a or C5a-des Arg tracheally in doses that caused dramatic pathologic responses resulting in acute respiratory distress or death within minutes [9]. However, with one exception [6] systema- tic investigations evaluating the effect of purified C5a administered intravenously do not exist. Therefore, we established the above described guinea-pig in vivo model.

The dose of 1.5 pg C5a kg- body weight caused no clinical signs detectable by sole observation of the whole animal’s behaviour. Yet measurements of lung physiology revealed significant effects: resistance increased to 140% paralleling a reduction of dynamic

506 T. HOFFMANN et ul.

compliance to 65%. The increase of breathing rate to 160% may be regarded as reflectory (control values before C5a injection were set to 100% in each case). These reactions may be perceived as shortness of breath, chest tightness or angina as reported by patients at the onset of shock or undergoing hemodia- lysis [20]. However, we cannot distinguish between a direct C5a effect on bronchial function and effects of accumulated PMNs (via C5a) as reason for the decreased lung function within the first 2 rnin after stimulus application. Because the single dose of C5a does not induce lung structure derangements but only reversible effects, it can be concluded that at an early stage of this potential ARDS model the observed lung dysfunction is not due to changes in lung structure, but to changes of bronchial function.

Timed from the end of a 30 s infusion of 1.5 pg kg-' C5a, PMN count fell to a nadir within 1 min demon- strating that the neutropenia-inducing activity which had been observed by several other workers employing more crude materials [7, 211, can clearly be related to this anaphylatoxin. To investigate where the granulo- cytes were left we performed histologic studies of the lung. The central vein catheter enabled us to infuse a formalin solution directly into the right heart of the deep anaesthetized animal and so to fix the lung situation at distinct moments after C5a infusion. Nevertheless, it cannot be excluded that those granulo- cytes which adhered only weakly in the capillaries were rinsed out by the 30 min lasting fixation procedure, in the course of which about 300 ml of formalin solution flowed through the vessels of the lung. This possibility is strengthened by our investigations with labelled PMNs, in which a great part of radioactivity that had been lost upon stimulus returned to circulation within minutes. So the number of PMNs found in the vascular bed might even be underestimated. Yet one minute after C5a infusion an average of 40 PMNs per 100 endothelial cells was counted. Compared with 14 PMNs/100 endothelial cells of control animals this means a nearly three-fold increase and agrees with the data of Redl et a/. [22] who also reported an almost three-fold augmentation of granulocyte-associated radioactivity in lung biopsies after traumatic shock in dogs. During the following hours the PMN-counts in the lung declined slowly and reached control values after 24 h.

At each time point nearly all observed PMNs were accumulated in the vasculature, and there was no evidence of migration towards the interstitial space or the alveolus (Fig. 6). This coincides with the results of Henson et al. [23] who injected CVF i.v. into rabbits On the other hand, Till et ul. [8], who administered CVF i.v. to rats, observed structural derangements of the capillary architecture, mainly damage of endothe- lial cells and of the vascular basement membrane. No such alterations could be detected by us. The most likely explanation for this difference might be that these authors generated a dose of C5a which was about one hundred times higher than the dose applied here,

and possibly other factors also generated by CVF, e.g. C3a, might have exerted an additional effect. The smooth muscle contraction of bronchioles and arter- ioles as reported by Stimler ef al. [9], who instilled AT- peptides tracheally, was not detected in this study. This may be due to the lower dose used here, and the different route of administration.

Our results are in accordance with those of Webster et al. [6], who injected partially purified C5a in rabbits and described a rapid neutropenia with following neutrophilia. In histological lung sections of animals killed 4 h after C5a administration they likewise discovered increased numbers of neutrophils without signs of tissue injury.

In contrast to Larson and co-workers [ 101, who also instilled AT-peptides tracheally and who found the leukocyte attracting activity of C5a-des Arg to be much higher than that of C5a, we noticed no differ- ences between the two peptides concerning their potency in inducing the disappearance of granulocytes from circulation. This is nonetheless, no contradiction because Larson's model differs widely from ours and a direct comparison of the results is impossible.

The molecular basis of the granulocyte sticking in the lung is yet under investigation. Alteration of

Figure 6. Histologic lung sections (Magnification x 1850). Upper part (a), control lung after injection of saline; lower part (b), condition of the lung 1 rnin after CSa-injection, showing granulocyte accumulation. After I min, 30 and 60 min difference of relative PMN content to control is highly significant (P<O.OI), after 90 min i t is significant (PcO.05) . After 180 min and 24 h number of PMNs is not significantly higher than in the control group.

THE ROLE O F C5a IN EARLY STAGES O F ARDS SO7

negative cell-surface charge and the expression of a surface glycoprotein heterodimer, called Mo 1, seem to be involved in granulocyte adhesiveness and aggrega- tion [24, 251. The questions whether cell-aggregation and cell-surface adherence represent functional equi- valents or not as recently proposed by Fehr et al. [26] and whether PMNs firstly aggregate and secondly embolize in the lung or primarily adhere to endothelial cells seems to be of less importance in our model. Both mechanisms lead to granulocyte-sticking in the capil- laries, a prerequisite for damaging the microvascula- ture by lysosomal enzymes and metabolites of oxida- tive burst and arachidonic acid, which are released, induced by C5a and CSa-des Arg (27).

To clarify the fate of sequestered granulocytes, studies with 5 1-chromium-labelled cells were carried out. After C5a infusion a drop of radioactivity in the blood paralleling the drop of granulocyte count occurred. Thereafter likewise a rapid increase up to three-quarters of the preinfusion value was detected indicating that most of the sequestered cells returned to circulation but one-quarter remained sequestered for about I h, which is consistent with the results of Brubaker and Nolph [28], who studied the fate of neutrophils during hemodialysis. After 1 h there was no further difference to values projected from the control period before C5a infusion. The 5 1 -chromium- labelled cells were judged to be viable to more than 97% by trypan blue-exclusion, and showed in all experiments an exponential decrease in the circulation with half times agreeing with those of earlier reports [29]. Thus, there were no indications that the beha- viour of the granulocytes might have been altered by the method of labelling. Moreover, no activity could be detected in the serum, excluding a release into circulation. Concerning the cells that caused the rebound of the granulocyte count our results point to two sources: firstly, a release from bone marrow as indicated by the banded cells and secondly, cells already in circulation prior to CSa injection, which were sequestered upon stimulus but returned to circu- lation within minutes. A mobilization of cells from the marginated pool is unlikely, since this is not indicated by our studies with labelled cells and moreover C5a is known to exert an opposite effect, i.e. i t increases adherence. The factors responsible for the neutrophilia are not yet known exactly. A direct stimulation of the bone marrow by C5a cannot be excluded. Others proposed a factor to be formed during neutropenia [30] and further studies will be necessary to clarify this question.

The potency of CSa to cause guinea-pig-platelet aggregation and liberation of their serotonin is well established in vitro [12, 311. However, the effects of systemically applied C5a on platelets in viuo are scarcely explored. In our model platelets also reacted with a drop that came slightly (1-2 min) after the nadir of the granulocytes. In contrast to the granulocytes the rebound was delayed and did not significantly exceed control values. The temporary thrombopenia is sug-

gested to be caused by C5a induced aggregation. This is supported by the finding of platelet-aggregates in lung capillaries after C5a application [5 ] . These aggre- gates were not obvious in our histologic sections, but this may be due to the different intravascular fixation- technique. A possible contribution of other mediators to aggregation, e.g. PAF, liberated from stimulated neutrophils, can be ruled out by the experiments with C5a-des Arg, in which the neutrophils reacted as well but not the platelets.

The differing behaviour of platelets in contrast to granulocytes, a fact we have already established in oitro [12], has to be discussed in the lights of a recent review on the C5a receptor [32]. Two possibilities are feasible: (i) there exist two different receptors for C5a and C5a- des Arg. Whereas granulocytes own both, platelets only possess the receptor for C5a; (ii) there is only one receptor on granulocytes reacting with both C5a and C5a-des Arg. But in this case the receptor on platelets for C5a must be different.

Our investigations were performed with intrave- nously administered, purified C5a in a dose range transiently producing signs of lung dysfunction. But, whether these changes reflect the respiratory symp- toms of patients during the onset of ARDS has to be further investigated. Moreover, C5a alone adminis- tered in a single dose seems to be insufficient to produce irreversible lung injury. By means of the presented model the next point of interest will be to investigate the parameters of a prolonged infusion of CSa, for several hours or days. The essential role of CSa was recently underlined by the prevention of a sepsis- induced ARDS in primates by anti-C5a antibodies 1331. The influence of additional factors which may contribute to this severe organ failure was underlined by a recent report [34]. These authors stress the importance of hypoxia in combination with comple- ment activation. It is evident from these studies that further mechanisms and factors working together with C5a or subsequent to C5a do exist in vivo as well, as there may exist pathomechanisms leading to ARDS totally independent of complement.

Acknowledgments The expert technical assistance of Ms. G. Riethmeister and of Ms. Messner is gratefully acknowledged. The authors also wish to thank Dr. A. Fiigner, Boehringer Ingelheim, FRG, in whose laboratory lung function was measured, and Dr. R. H. Boedeker from the Institut of Medical Informatics, University of Giessen, for his advice in statistical analysis. The work was supported by a grant of the Deutsche Forschungsge- meinschaft, SFB 31 1, D4 Mainz.

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