complement activated granulocytesdownloads.hindawi.com/journals/mi/1992/246976.pdf · acute...

Research Paper

Mediators of Inflammation, 1, 177-181 (1992)

ACTIVATION of polymorphonuclear granulocytes (PMNs)by Cha is thought to be important in the pathogenesis ofmultiple organ failure during sepsis and after trauma. Inour experiment exposure of human PMNs to autologouszymosan activated plasma (ZAP) leads to a rapid increasein chemiluminescence. Heating the ZAP at 56C for 30rain did not alter the changes, while untreated plasmainduced only baseline activity. The respiratory burst couldbe completely abolished by decomplementation and pre-incubation with rabbit antihuman Cha antibodies. Ob-servation of human omentum using electron microscopyshowed intravascular aggregation of PMNs, with capillarythrombosis and diapedesis of the cells through endothelialjunctions 90 s after exposure to ZAP. PMNs caused disrup-tion of connections between the mesothelial cells. After4 rain the mesothelium was completely destroyed, andconnective tissue and fat cells exposed. Native plasma andminimum essential medium did not induce any morpho-logical changes. These data support the concept that Chaactivated PMNs can cause endothelial and mesothelialdamage in man. Even though a causal relationship be-tween anaphylatoxins and organ failure cannot be provedby these experiments Cha seems to be an important medi-ator in the pathogenesis of changes induced by severe

sepsis and trauma in man.

Key words: ARDS, Cha, Complement activated granulocytes,Mesothelium, Oxygen free radicals, Sepsis

Complement activatedgranulocytes can causeautologous tissue destructionin man

E. Lhde,1"cA H. Raude, M. LLick,E. Kraas and W. Lierse2

1Surgical Department, Academic HospitalMoabit, Turmstr. 21, D-1000 Berlin 21, Germany;21nstitut of Neuroanatomy, University ofHamburg, Martinistr 52, 2000 Hamburg 20,Germany

ca Corresponding Author

Introduction

Systemic complement activation plays a key rolein acute organ dysfunction during sepsis. 1-3 It hasbeen demonstrated in several animal models thatthe lung is one of the main targets of activatedcomplement. Histological changes consist ofpulmonary haemorrhage, interstitial oedema, en-dothelial damage and leucocyte plugging.4-6

These changes observed in animal modelsparallel the findings in patients with severe sepsisas well as after trauma and cardiopulmonarybypass,2’7 are generally designated as adult respir-atory distress syndrome (ARDS) independent oftheir aetiology. Cha has been considered a potentand important mediator of these changes. Its targetis a 47kD receptor on the cell surface ofpolymorphonuclear granulocytes (PMNs). Chamediates the release of free oxygen radicals,adherence, aggregation and chemotactic migrationof these cells. These changes may contribute to thedevelopment of capillary leakage observed duringacute illness.

However, there is a lack of informationconcerning the actual mechanism of the reaction ofPMNs to activated complement in human tissue.For example, it is still unclear whether, and to what

1992 Rapid Communications of Oxford Ltd

extent, the in vitro observed Cha mediated release oftoxic oxygen radicals causes damage to human cellsin vivo. Therefore, we used human peritoneum to

investigate the effects of activated complement onautologous human tissue.

Methods

Cells: Heparinized blood was obtained from healthyadult volunteers. PMNs were separated by Ficolldensity gradient and the remaining red cells lysedwith distilled water/1.8% saline solution. PMNswere then washed twice in minimum essentialmedium (MEM) (Dulbecco pH 7.4 without phenolred and bicarbonate) and resuspended at a

concentration of 5-6 x 103 cells//l in MEM.Pappenheim staining showed that 98% were PMNsand trypan blue exclusion showed 94-96% of cellsto be viable.

Plasma: Plasma was obtained from heparinizedblood from human volunteers and kept in storageat 4C for a maximum of 6 h. Complement wasactivated by incubating the plasma with boiledzymosan (Sigma Chemical Co., St Louis, MO) at

37C for 30min followed by centrifugation at

2000 g. The supernatant was stored at 4C for a

Mediators of Inflammation. Vol 1992 177

E. L6hde et al.

maximum of 6 h. Decomplemented plasma wasprepared by heating the plasma to 56C for 30 min.C5a inhibition was obtained by preincubation ofZAP with rabbit immunoglobulins cross-reactingto human C5a (DAKO code A O 55 Boehringer-Ingelheim, Germany) at 37C for 15 min.

Chemiluminescence: Chemiluminescence was measuredin a Biolumat LB 9500 and LB 922 (Berthold Mod.210) and data analysis was performed by a HewlettPackard 97S computer. Isolated PMNs wereincubated with Luminol (4-amino-2,3 dihydroxy-1,4-phthalazinedione, Sigma Chemical Co., StLouis, MO) for detection of reactive oxygencompounds. When baseline activity was reachedautologous plasma was added to give a finalconcentration of 32%. The generation of reactiveoxygen compounds was continuously measured andcomputed as the maximal count every 30 s.

Scanning electron microscopy: Heparinized blood wasobtained from patients scheduled for chole-cystectomy and prepared as mentioned above. Partof the greater omentum was carefully removedimmediately after opening the abdomen. Tissuespecimens were exposed to autologous ZAP andnative plasma as well as to MEM with and withoutthe addition of autologous PMN for 0, 1.5, 4, and25 min. The experiment was commenced within 15min after surgical removal of the tissue, which was

kept humidified throughout. After termination ofexposure the specimen was fixed in 2.2%glutaraldehyde followed by osmium exposure.Dehydration was carried out with alcohol inincreasing concentrations and exchange to freon.Critical point drying and gold coating was

performed in an Edwards sputter coater for 2.5 minand the specimens were evaluated in a Novoscan30 (Zeiss).The experiment was approved by the human

experimentation committee, and all patients partici-pating gave informed consent.

Results

Exposure of PMNs to autologous zymosanactivated plasma caused a rapid increase ofchemiluminescence due to an accelerated generationof free oxygen radicals (Figure 1). The increasecould already be detected after 30 s and reached itsmaximum after 5-7 min. Zymosan activity waspreserved even after heating the activated plasma at56C for 30 min and the PMN showed an identicalpattern of free oxygen generation. Native plasmainduced only a baseline activity and decomple-mentation of the plasma prior to zymosanincubation abolished the generation of plasmaactivity completely. The respiratory burst of PMN

x 103

counts/min

70--

30--

Io (3)

0 2 3 4 5 6 li

FIG. 1. Chemiluminescence measurement of isolated human PMNs inautologous plasma. The exposure to ZAP (curve as well as heated ZAP(curve 2) induces an increase generation of oxygen derived free radicalsfrom the cells after 30 s. Native plasma (curve 3) does not result inincreased metabolic activity. Plasma heating prior to zymosan incubationabolishes the generation of active complement fragments (curve 4).

secondary to exposure to ZAP could be readilyinhibited by dose-dependent incubation of ZAPwith cross-reacting rabbit anti-human C5 antibodies(Figure 2).A scanning electromicrograph of the normal

greater omentum of man is shown in Figure 3a.The surface is formed by a monolayer ofpolygonal mesothelial cells with microvilli. Theyare densely interconnected and overlay connectivetissue and fat cells. In addition, numeroussuperficial vessels can be seen.

Within 90 s after ZAP exposure PMNs appear onthe mesothelial surface presumably due to directedmigration. Around the PMNs the mesothelial cellsretract, the interconnections between these cellsbreak up and gaps appear between the formerlytightly packed cells (Figure 3b).

Four minutes after ZAP exposure the me-sothelium in the vicinity of the cells is completelydestroyed. The submesothelial connective tissue isnow only partially covered by the remainingdamaged mesothelial cells (Figure 3c). In these areaseven fat cells are exposed while regions more distantto the PMNs remain morphologically intact.Exposure of the omentum to native plasma orMEM does not induce any changes.A closer examination of the superficial capillaries

reveals that diapedisis of PMNs through the vesselwall begins within 90 s after ZAP exposure. Figure

178 Mediators of Inflammation. Vol 1992

Activated granulocytes and tissue destruction

counts/min

140

x 103

120

100

80--

60--

40--

20--

2 10 Vol %

C5-antibody

FIG. 2. The ZAP-mediated generation of 02-species from isolated PMNsshown as an integral of the first 4 min after exposure. Prior incubationof ZAP with cross-reacting mouse anti-human C5/C5a antibodies. IgGcauses a marked CL-reduction up to 87% of the mean.

FIG. 3a. Scanning electron microscopy of native human greater omentum.Mesothelial cells form a tight monolayer at the surface. The gross tissuestructure is formed by underlying fat cells. Superficial capillaries aredemonstrated.

4 shows PMNs during the migration through theendothelial junctions of the capillaries. Simultan-eously, the formation of intravascular PMNaggregates is observed (Figure 5). The PMNsadhere tightly to each other, the capillary flow isinterrupted and the endothelium in the region ofthe micro-aggregates destroyed. Neither nativeplasma nor MEM leads to a similar phenomenon.

Discussion

One essential feature of the interaction of C5awith PMNs seems to be the great speed of reaction.It is due to the small size of the molecule and itshigh aflqnity to specific receptors on the surface ofPMNs. Exposure to C5a leads to a 50% saturationof the receptors within 1 min.8

The data presented here show that the presenceof active complement fragments can initiate anactivation of PMNs which, in turn, causes intensedamage to autologous tissue. After the complementcascade has been activated, foreign antigens (e.g.bacteria) are not necessary for the activation ofeffector cells. The developing cytotoxicity isundirected but clearly restricted to the immediateenvironment of the PMNs. More distant tissue areas

FIG. 3b. 90s after ZAP exposure. Retraction and disconnection ofmesothelial cells in the near environment of activated leucocytes arevisible as arrowed. The leucocytes themselves appeared at the tissuesurface by their own chemotactic locomotion.

....iiiii@!i::> i#....,:<ii

"’..:i;iiiiiii!i!ii::..i!.i. {{iiiig

FIG. 3c. 3 min after ZAP exposure. The mesothelial cell layer has beendestroyed. The cross network of submesothelial connective tissue andunderlying fat cells are uncovered as arrowed.

Mediators of Inflammation. Vol 1992 179

E. Lb’hde et al.

FIG. 4.90 after ZAP exposure. Intravascular leucocytes as arrowed havebeen chemotactically activated and are migrating through endothelialgaps out of the capillaries.

remain morphologically intact. Thus, activatedcomplement does not induce morphologicallydetectable changes of human mesothelial cells byitself. The damage is rather dependent on theactivation of PMNs.The cytotoxicity of C5a activated PMNs to

homologous endothelial cells in vitro was firstdemonstrated by Sacks. The release of free oxygenradicals leads to the oxidation of the unsaturatedfatty acids of the phospholipids in the cell wall. Ourdata also show a close correlation between themesothelial damage and the kinetics of the oxygenrelease by PMNs measured by chemiluminescence.A maximal increase of oxygen radical productionof isolated PMNs occurred within a few minutesafter ZAP exposure. Scanning electron microscopyrevealed extensive tissue destruction after the sametime interval. A similar temporary relationship wasobserved by Goldstein et al.l In their experimentsmore than 80% of the maximum obtainable freeoxygen radicals derived from PMNs stimulated byisolated C5a, as measured by cytochrome reduction,were generated within the first 10 min. The actualintensity of the respiratory burst of PMNs in vivo

might even be considerably higher because surfacecontact has been thought to promote oxygen radicalrelease. 11 Data from animal experiments show thatthe addition of dismutase or catalase serving as

oxygen radical scavengers as well as granulocytedepletion can provide significant protection againstC5a mediated tissue damage.6

The data presented here also confirm earlierobservations about the chemotactic migration ofPMNs. It is known that C5a as a physiologicalmediator of inflammation induces chemotacticmigration of PMNs.12’3 However, in spite of therapid activation of PMNs by C5a a significant effecton directed migration can only be detected after 60min. 12 Nevertheless, morphological studies of theZAP activated PMNs reveal the formation ofpseudopodia and membrane ruflqing within seconds

180 Mediators of Inflammation. Vol 1992

FIG. 5.90 after ZAP exposure. Some intravascular activated leucocytesform tight leucoaggregates within the vessels. Subsequently thesurrounding endothelium is destroyed and the capillary flow interrupted.

after exposure. 14’1s Other alterations of thecytostructure consistent with the initial phase ofdirected cellular migration have also been detectedwithin seconds of exposure.

Our data show that the chemotactic response ofPMNs to C5a in vivo occurs within seconds andfollows the same time course as the other C5arelated effects. Intervascular PMNs become rapidlyactivated by C5a and pass through the endothelialjunctions of the capillaries into the surroundingtissue. The rapidity of C5a diffusion into thecapillaries, binding to the receptors, induction ofthe cellular response and directed migration out ofthe vessel emphasizes the potency of thisanaphylatoxin in vivo.One of the main morphological features which

correlates to the physiological changes of adultrespiratory distress syndrome (ARDS) is theformation of microemboli in the pulmonarycapillaries by aggregated PMNs. Earlier studies ofisolated human PMNs, using electron microscopy,showed the development of tight cell-to-cellaggregations after exposure to ZAP serum. 14’15

Similar functional studies by aggregometry demon-strated a maximal cell aggregation 90 s after ZAPexposure. TM The mechanism of aggregation isthought to be a reduction of the surface charge of

16the granulocytes after binding of C5a to receptors.Our observations confirm that activated comple-

ment can lead to the formation of intervascularleucocyte aggregates in human capillaries. Thromb-osis of the capillaries could be observed after 90 s,the endothelium was destroyed and vascular flowinterrupted. Thus, not only human mesotheliumbut also human endothelium is susceptible to theC5a induced cytotoxicity of PMNs. The con-

sequences of these changes in capillary flow aretransudation, haemorrhage and hypoxaemia. The

Activated granulocytes and tissue destruction

microvascular structure of the lungs is consideredespecially vulnerable to the cytotoxic effect ofcellular aggregates.4’5 In fact only 10-15 cells seem

to be suflqcient for the creation of microvasculardestruction within seconds, explaining the rapidphysiological effect of leucocyte sequestrationcaused by systemic complement activation in man.

In summary, the findings presented may wellsupport the concept that C5a activated PMNs playa key role in the pathogenesis of capillary damagein man as it is found in ARDS. Because of our lackof knowledge concerning the regulatory mechan-isms of the inflammatory reseponse it would bepremature to interpret the data as proving a causalrelationship between anaphylatoxins and organfailure. Nevertheless, the complement derivedcleavage product C5a can be considered a highlyactive principle in the pathogenesis of the changesinduced by severe sepsis and trauma in man.

References1. Hammerschmidt DE, Weaver I,J, Hudson LD, Craddock PR, Jacob HS.

Association of complement activation and elevated plasma-CSa with adult

respiratory distress syndrome. Lancet 1980; 1: 947-949.2. Jacob HS. Role of complement and granulocytes in septic shock. Acta Chir

Scand Suppl 1980; 499 97-106.3. Solomkin JS, Cotta LA, Satoh PS, Hurst JM, Nelson RD. Complement

activation and clearance in acute illness and injury: evidence for C5acell-directed mediator of the adult respiratory distress syndrome in

Surgery 1985; 97 66&-677.4. Hohn DC, Meyers AJ, Gherini ST, Beckmann A, Markinson RE, Churg

AM. Production of acute pulmonary injury by leukocytes and activatedcomplement. Surgery 1980; 88: 48-58.

5. Hosea S, Brown E, Hammer C, Frank M. Role of complement activation inmodel of adult respiratory distress syndrome. J Clin Invest 1980; {;6:

375-382.6. Till GO, Johnson KJ, Kunkel R, Ward PA. Intravascular activation of

complement and acute lung injury--dependency neutrophils and toxic

oxygen metabolites. J Clin Invest 1982; 69: 1126-1135.7. Jacob HS. Complement-mediated leucoembolization: mechanism of tissue

damage during extracorporeal perfusions, myocardial infarction and inshock--a review. Q j Med New Series LII 1983; 2{}’/: 289-296.

8. Chenoweth DE, Hugli TE. Human C5a and C5a analogs probes of theneutrophil C5a receptor. Molecular Immunol 1980; 1’/: 151-161.

9. Sacks T, Moldow CF, Craddock PR, Bowers TK, Jacobs HS. Oxygenradicals mediate endothelial cell damage by complement-stimulatedgranulocytes. J Clin Invest 1978; 61: 1161-1167.

10. Goldstein IM, Roos D, Kaplan HB, Weissmann G. Complement andimmunoglobulins stimulate superoxide production by human leucocytesindependently of phagocytosis. J Clin Invest 1975 56: 1155-1163.

11. Dahinden CA, Fehr G, Hugli TE. Role of cell surface contact in the kineticsof superoxide production by granulocytes. J Clin Invest 1983; ’/2: 113-121.

12. Fernandez HN, Henson PM, Otani A, Hugli TE. Chemotactic response to

human C3a and C5a anaphylatoxins. J Immuno11978; 12{}: 109-115.13. Hugli TE. The structural basis for anaphylatoxin and chemotactic functions

of C3a, C4a and C5a. CRC Critical Reviews in Immunol 1981 321-366.14. Craddock PR, Hammerschmidt D, White JG, Dalmasso AP, Jacob HS.

Complement (C5a)-induced granulocyte aggregation in vitro. J Clin Invest1977; 6{}: 260-264.

15. 1,6hde E, Kraas E, Abri O, I,ierse W. Activated complement--aof organ failure in necrotizing pancreatitis. In: Peiper H-J, ed. Chir. Forumf experim, klinische Forschung. Heidelberg: Springer-Verlag, 1987: 343-347.

16. Gallin JI, Durocher JR, Kaplan AP. Interaction of leucocyte chemotacticfactors with the cell surface. I. Chemotactic factor-induced changes ingranulocate surface charge. J Clin Invest 1975; 55: 1049-1057.

ACKNOWI,EDGEMENT. The authors wish to thank Mrs Susanne Feldhausand Mr Klaus Siebert for their excellent technical assistance and Mrs de Bourfor preparing the manuscript.

Received 5 March 1992"accepted 27 March 1992

Mediators of Inflammation-Vol 1992 181

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