Demonstration of a local exhaustion of complement components ...

Clin. exp. Immunol. (1980) 42, 506-514.

Demonstration of a local exhaustion ofcomplement componentsand of an enzymatic degradation ofimmunoglobulins in pleuralempyema: a possible factor favouring the persistence of local

bacterial infections

D. P. LEW, J.-P. DESPONT, L. H. PERRIN, M.-T. AGUADO, P. H. LAMBERT &F. A. WALDVOGEL Infectious Disease Division and the World Health Organization Immunology

Research and Training Center, Department ofMedicine, University ofGeneva, Switzerland

(Acceptedfor publication 13 June 1980)

SUMMARY

Local bacterial infections such as abscesses or purulent exudates most often containnumerous, easily culturable bacteria despite an intense inflammatory reaction character-ized by the ingress of polymorphonuclear leucocytes. In order to understand themechanisms leading to such a persistent infection, we used pleural empyema as a modeland measured the levels and catabolism of complement as well as of immunoglobulins in28 infectious pleural effusions associated with either a positive or with a negative bacterialculture. Classic and alternative pathway haemolytic activities, factor B and C4 haemolyticactivities as well as native C3 were markedly decreased or undetectable in most culture-positive effusions when compared to culture-negative effusions (P< 0 005); breakdownproducts of factor B and C3 were markedly increased in culture-positive fluids. Eleven outof 14 culture-positive fluids exhibited IgG breakdown as opposed to none of the culture-negative fluids. In seven out of 14 culture-positive fluids, incubation with 1251-IgG led totheir in vitro breakdown. This proteolytic activity could be abolished by preincubation ofthe culture-positive fluids with normal sera. Thus, increased catabolism of complementand breakdown of immunoglobulins, both leading to local consumption of immunereactants, could be one of the causes for bacterial persistence in pleural empyema.

INTRODUCTION

Abscesses and purulent exudates are characterized by local survival of bacteria despite the presenceof large numbers of polymorphonuclear leucocytes (PMNLs). The mechanisms leading to thisparadoxical situation have not yet been fully explained, but one of its practical consequences is theneed to drain most purulent collections in order to obtain cure (Sherman, Subramanian & Berger,1977).

It is generally accepted that complement, antibody and PMNLs are essential components indefences against extracellular pyogenic bacterial infections, to which patients with agranulocytosis(Bodey et al., 1966), agammaglobulinaemia (Rosen & Janeway, 1966) or C3 deficiency (Alper,Colten & Rosen, 1972; Alper, Bloch & Rosen, 1973) are highly susceptible. Complement and

This study was presented in part on 4 October 1978 at the Interscience Conference on Antimicrobial Agentsand Chemotherapy, Atlanta, Georgia.

Correspondence: F. A. Waldvogel, MD, Chief Infectious Disease Division, University Hospital, 1211Geneva 4, Switzerland.

0099-9104/80/1200-0506S02.00 C 1980 Blackwell Scientific Publications

5o6

Complement and immunoglobulins in empyema 507immunoglobulins, the main phagocytosis-promoting factors, can be altered during clinical orexperimental infections: thus, serum complement activation leading to decreased complementcomponents has been described during acute bacteraemia (Fearon et al., 1975), and several workershave demonstrated breakdown ofimmunoglobulins by bacterial orPMNL proteases (Waller, 1974;Plaut, Wistar & Capra, 1974). Little, however, is known about extravascular or interstitial fluidlevels of complement and immunoglobulins in the presence of infection. Recently, we have reporteda marked decrease in opsonic activity and in classical pathway-mediated haemolytic activity,coinciding with the appearance of C3 breakdown products in empyema fluids (Lew et al., 1979).These data suggested that continuous consumption of immune reactants could occur withininfected foci such as empyemas leading to an acquired local immune deficiency and hence topersistence of bacterial disease. The present study was therefore undertaken in order to determinethe levels and the catabolism of complement components, as well as of immunoglobulins presentlocally in pleural empyema, a paradigm of local, persistent bacterial infection. Evidence for localbreakdown of these essential antibacterial humoral components is presented here.

MATERIAL AND METHODS

Study population. Patients undergoing diagnostic thoracentesis at the Department of Medicineof the Geneva University Hospital were included prospectively in this study. During the period ofthe study 26 patients presented with a pleural effusion associated with clinical, bacteriological andradiological evidence of pneumonia. In two of these patients, fluid obtained from a secondthoracentesis performed after a 1-week interval was also studied.

Microbiological studies. All pleural effusions that were subsequently investigated were immed-iately examined microscopically and cultured on standard and anaerobic media. Micro-organismswere identified in the microbiology laboratory according to well-established techniques (Lennette,Spaulding & Truant, 1974). In 14 fluids, microscopical examination and cultures remained negativefor bacterial growth (culture-negative group), whereas the other 14 cultures yielded pathogenicorganisms (culture-positive group). This latter group encompassed six fluids yielding primaryanaerobic organisms, seven yielding aerobic organisms and one yielding mixed aerobic-anaerobicgrowth, and had all the macroscopic and cytological characteristics of empyema, as opposed toculture-negative effusions.

Collection ofsamples. All the samples were immediately mixed with EDTA (final concentration20 mmol/l) or kept in native form, centrifuged at 1 ,500g for 15 min; the supernatants were aliquotedand kept at - 70'C until used.

Complement studies. C4, factor B, native C3 and total C3 were measured by radial immunodiffu-sion using specific antisera (Perrin, Lambert & Miescher, 1975). Small molecular weight fragmentsof C3 and factor B, respectively C3d and Ba, were measured using a two-step immunochemicalprocedure (Lennette et al., 1974). Classical pathway-dependent haemolytic activity (CH50) wasstudied using a continuous-flow system (Lennette et al., 1974). Alternative pathway-dependentopsonic activity was assessed by measuring the ingestion rates of endotoxin-coated paraffinparticles by normal polymorphonuclear leucocytes (PMNLs) as described previously (Stossel,1973). Haemolytic C4 assay was performed using C4-deficient guinea-pig plasma and measuring thehaemolysis of sheep red cells. Haemolytic alternative pathway activity was measured by functionalhaemolytic assay using a modification of the technique of Platts-Mills & Ishizaka (1974). Briefly,serial serum dilutions were mixed with a constant number of 5'Cr-labelled rabbit red cells andincubated at 37°C for 60 min; lysis was measured by counting radioactivity of the supernatant aftercentrifugation. Factor B haemolytic activity was similarly measured after addition of a constantamount of factor B-depleted serum to each serum dilution.

Immunoglobulin studies. Quantitation of IgG, IgA and IgM was performed with standardcommercial radial immunodiffusion kits (Behring Diagnostics, Somerville, New Jersey), and by anephelometric method using a Technicon autoanalyser. Studies on immunoglobulins and theirfragments were performed with various procedures. First, immunoelectrophoresis was carried outusing rabbit antisera directed against human IgG, Fc and Fab. Secondly, material precipitated by

ammonium sulphate 50% was studied in 10% polyacrylamide gel electrophoresis in the presence ofsodium dodecyl sulphate. Thirdly, density-gradient studies of pleural fluid specimens were per-formed by adding 0 5 ml of pleural fluid or control sera to linear 10-40% (w/v) sucrose gradients in01 M borate NaCl buffer; after ultracentrifugation at 50,000g for 4 to 18 hr serial gradient fractionsof 250 p1 were collected for subsequent analysis and determination of optical density (OD) at 280nm.

Proteolytic activity of pleuralfluids on IgG substrate. For these experiments human IgG waspurified by DEAE chromatography and then labelled with 1251 according to McConahey & Dixon(1966). For the demonstration of IgG proteolysis, 10 pl of a 1251-IgG preparation (2 mg/ml) wereadded to 25 pl of pleural fluid diluted in 40 pl of isotonic saline and the mixtures incubated at 370Cfor 4 hr; thereafter, 50 p1 of normal human serum were added, followed by 2 ml of 40% ammoniumsulphate. After a 30-min incubation at 40C, the tubes were centrifuged at 2,00Og for 20 min and theradioactivity determined in the pellet. Results were expressed as per cent decrease in IgG precipi-tated by ammonium sulphate [(per cent IgG precipitated following incubation with normalserum - per cent IgG precipitated following incubation with the pleural fluid sample)/(per cent IgGprecipitated following incubation with normal serum)].

Statistical evaluation. For significance of the results of the two patient populations, the unidirec-tional Wilcoxon test was applied. For correlation analysis, the Spearman correlation coefficientswere used.

RESULTS

Complement studiesVery low values of classical pathway-dependent haemolytic activity (CH50) were observed inpleural fluid samples from patients with culture-positive pleural effusions when compared withculture-negative infectious fluids (Fig. la) (P<0005). In order to assess alternative pathwayfunction, two different assays partly depending on the integrity of this system were used: first, thehaemolysis of rabbit red cells (Fig. la) and second, the ability ofthese fluids to promote ingestion bynormal PMNLs of endotoxin-coated paraffin particles (Table 1). Culture-positive fluids exhibitedvery low activities as measured by both assays (P <0 005) and a significant correlation was foundbetween the values obtained with these two techniques for all fluids tested (r = 0-6740, P < 0-0 1).

Factor B and C3 in pleural fluids were first evaluated by functional haemolytic assays. Culture-positive fluids exhibited lower levels of factor B and C3 haemolytic activities when compared withculture-negative fluids (P <0005) (Fig. lb, Table 1). In contrast, factor B protein and C4 protein,measured immunochemically, exhibited similar levels in culture-positive and culture-negative

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culture-positive (Culture pos.) and culture negative (Culture neg.) infectious pleural effusions. (b) Factor Bhaemnolytic activity (Fact. B haem), factor B protein (Fact B tot.) and Ba fragments in culture-positive andculture-negative infectious pleural effusions. The results are expressed as a percentage of reference preparation.

5o8 D. P. Lew et al.

Complement and immunoglobulins in empyema 509Table 1. Complement components + immunoglobulin levels and their respective breakdown products in infec-tious pleural fluids*

Culture-positive Culture-negative Statistical evaluation(P value)

Alternative pathway-mediatedopsonic activity (%)t 41 + 34 470 + 582 <0001

Total C4 (%)+ 46 0 + 34 56 0 + 48-0 n.s.Haemolytic C4 () 3-0 + 7 260 + 29.0 < 0-05Total C3 (%)I 66-0 + 46 57 0+44 0 n.s.Native C3 (%)+ 6 0+ 14 38-0+23.0 <0 005C3d fragment (%)§ 50-0+44 260±+ 24 0 < 0 05

Total IgG (mg/100 ml) 425.0 + 320 7 711 0+ 210.3 < 0 05Total IgA (mg/100 ml) 175 0+ 146 0 159 0+ 104.0 n.s.Total IgM (mg/ 100 ml) 40 0+ 24-0 64 0+ 25 0 <0 01IgG breakdown products" 11/14 0/14Protein concentration (g/l) 40 0+ 11-0 42 0+ 8-0 n.s.

* Data presented as mean + s.d.t Measured by uptake ofendotoxin-coated paraffin particles and expressed as a percent-

age of values found with normal sera pool.+ Expressed as a percentage of values found with normal plasma pool.§ Expressed as per cent normal pool sera activated at 37 C for 60 min by inulin (2 mg/ml).WT Number of fluids in which there were multiple precipitation lines in immunoelectro-

phoresis using specific anti-IgG antisera.n.s. =Difference not significant.

fluids. The levels of fragment Ba, the small fragment of factor B, were markedly increased inculture-positive fluids (P < 0 005). The ratio of mean fragment Ba concentration to mean haemoly-tic factor B was markedly elevated in culture-positive fluids (3 4) when compared with culture-nega-tive fluids (0 4).

C3 concentrations were measured by using antisera against either native C3 or C3c in order toevaluate the proportion of C3 that had undergone structural changes leading to the loss of thenative antigen (Table 1). Significantly lower levels ofC3 were found in culture-positive fluids when anative C3 antiserum was used. When using anti-C3c antisera that reacts with native C3, C3b andC3c, no significant difference between the two groups was found.

Levels of C3d, the small breakdown product of C3, were higher in culture-positive fluids (Table1). Thus, the ratio of mean C3d to mean native C3 was much higher in culture-positive fluids (3 8)when compared to culture-negative fluids (0-7). No significant correlation was found between levelsof C3d and Ba fragments in all pleural fluids tested.

Immunoglobulin studiesWhen immunoelectrophoresis (IEP) was carried out using specific anti-IgG, Fab and Fc antisera,eleven out of 14 culture-positive fluids exhibited evidence of IgG breakdown into Fab- and Fc-likefragments: a representative example ofa culture-positive fluid is shown in Fig. 2. By contrast no IgGbreakdown could be detected in culture-negative fluids. Further evidence for IgG breakdown inempyema samples was obtained by ultracentrifugation studies using sucrose density-gradients.Gradient fractions were analysed for their IgG content by radial immunodiffusion and by nephelo-metry. In several cases most of the IgG antigenic determinants (Fcy) appeared in fractions smallerthan 7S while only a small fraction of intact 7S IgG was detectable (Fig. 3).

The analysis ofSDS polyacrylamide gel electrophoresis pattern (after reduction and alkylation)of the material precipitated from culture-negative pleural fluids by 50% saturated ammoniumsulphate demonstrated the presence of polypeptide chains with a molecular weight similar to heavy

D. P. Lew et al.

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Fig. 2. Immunoelectrophoresis ofculture-positive and culture-negative pleural fluids. (1) Culture-positive fluid;(2) culture-negative fluid; (3) normal human serum. IgG= Rabbit anti-human IgG antiserum; Fc= rabbitanti-human Fc antiserum; Fab= rabbit anti-human Fab antiserum. In culture-positive fluid (1), anti-IgGantiserum reveals the presence of two lines of precipitation with different electrophoretic mobilities. Thecathodal line reacts mostly with an anti-Fab antiserum and the anodal line reacts with an anti-Fc antiserum. Inculture-negative fluid (2) and in normal human serum (3), anti-IgG, anti-Fab and anti-Fc antisera reveal a singleline of precipitation with the typical electrophoretic mobility of intact IgG.

(H) and light (L) chains of IgG. By contrast, in culture-positive fluids only low molecular weightmaterial was visible on the gel (Fig. 4). As shown in Table 1, although protein concentrations were

similar, IgG and IgM concentrations were significantly lower in culture-positive fluids whereas no

difference in IgA concentration was found between the two groups of fluids. Finally, IgG and IgMlevels were found to be similar in the serum samples of both groups of patients.

Since these results suggested breakdown of immunoglobulins in empyema as demonstrated for

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Fig. 3. Normal human immunoglobulins (o-o) and pleural effusions (culture-positive [o --- ol and culture-negative [v-.]) were applied on three linear sucrose density-gradients. After ultracentrifugation fractionswere collected and the amount of IgG present on each of them is reported as per cent of the total amount appliedon the gradient.

510

Complement and immunoglobulins in empyema

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Fd~ Fc

Fig. 4. SDS polyacrylamide gel electrophoresis (l0O%) of material precipitated by ammonium sulphate frompleural fluids. (1) Reduced F(ab')2 fragments purified from normal IgG. (2) Reduced culture-positive materialprecipitated by ammonium sulphate. (3) Reduced culture-negative material precipitated by ammonium sul-phate. (4) Reduced Fc fragments purified from normal 1gG. In culture-negative fluid (3), proteins with amolecular weight similar to heavy (H) and light chains (L) are clearly present. By contrast, in culture-positivefluid (2) only low molecular weight material is visible on the gel.

complement components, the products of incubation (370C, 4 hr) of 10 P1 251-IgG (2 mg/ml) witheither 25 P1 of a culture-positive or 25 P1 of a culture-negative pleural fluid were examined by sucrosedensity-gradient ultracentrifugation analysis (Fig. 5). Radioactivity in the culture-positive fluid-i251-IgG mixtures -was maximal in the small molecular weight gradient fractions, whereas almostnone could be detected in the 7S fraction suggesting extensive IgG breakdown into small fragments.By contrast, culture-negative fluid 251-IgG mixtures -displayed maximal radioactivity in the 7Sgradient fractions as found in control sera, demonstrating that added 1251-IgG had remained intactin these samples.

The quantitation of IgG proteolytic activity of all the pleural fluids was achieved by incubatingthe 14 culture-positive infectious fluids and the 14 culture-negative infectious fluids with 1251I-IgG at37 C for 4 hr and by precipitation of the residual IgG by addition of 4000 ammonium sulphate. Atthis concentration, ammonium sulphate was found to precipitate intact IgG and large IgGfragments but not its smaller fragments. Seven of 14 culture-positive fluid incubations led to amarked decrease in the amount of '251-IgG precipitated (Fig. 6); none of the culture-negative fluidincubations caused any significant decrease in the amount of precipitable '251-IgG; the samenegative results were obtained with control sera. A highly significant inverse correlation betweenproteolytic activity (measured as decrease of 1251-IgG precipitable) and IgG levels was found(r =0872, P <0005) in the culture-positive fluids. Finally, samples of infectious pleural effusionswere preincubated at 370C for 1 hr with various human sera. Proteolytic activity of the variousfluids was expressed as per cent decrease of precipitable 1251-IgG measured on the 60-mmn samples

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512 D.P.Lewet al.

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Fig. 5. 1251 measured in sucrose gradient fractions obtained after preincubation for 4 hr at 37TC with aculture-positive fluid (A a), a culture-negative fluid (o---o) and normal human serum (---- *). Nodifference in sedimentation velocity was observed between labelled IgG incubated with culture-negative fluidand normal human serum. In contrast, labelled IgG incubated with culture-positive fluid was degraded intosmall fragments.

Fig. 6. 1251-IgG precipitable by ammonium sulphate 40% after incubation for 4 hr at 37 C with culture-positivefluids and culture-negative fluids. Results are expressed as (per cent IgG precipitated after incubation withnormal serum -per cent IgG precipitated following incubation with pleural fluid)/(per cent IgG precipitatedfollowing incubation with normal serum).

when compared to those obtained at time 0 (mean of three samples each). Proteolytic activity inempyemas decreased from 83-8 + 12 7 (after 60 min) to 6 2 + 11 8 when incubated in the presence ofpooled sera; the values of protpolytic activity were also sharply decreased when the empyema fluidswere incubated in the presence of autologous sera (19.5+6 3) and agammaglobulinaemic sera(317 + 13 1). By contrast, empyema fluid incubated with albumin at the same concentrationshowed only a slight decrease in proteolytic activity (69 7 + 13 5). These results suggest that freshhuman serum has the capacity to inhibit to a great extent the breakdown of immunoglobulins inempyema.

DISCUSSION

The results presented in this study provide evidence in favour of an increased catabolism of certaincomplement components and immunoglobulins, leading to a consumption of these opsonic factorsin pleural empyema. Classical pathway- and alternative pathway-dependent haemolytic activities,factor B and C4 haemolytic activities as well as native C3 were all found to be almost undetectable inmost pleural effusions associated with a positive bacterial culture, in contrast to the values obtainedin culture-negative pleural effusions. In addition, evidence for local breakdown of B and C3 was

shown by the direct quantitation of Ba and C3d fragments suggesting that in culture-positivepleural effusions, low residual complement values are due to an inappropriately high breakdown ofsome of its factors. Since these very low values were found almost exclusively in pleural effusionswith heavy bacterial concentrations, as demonstrated by Gram stain and bacterial cultures, it can beconcluded that in empyema, very little functional complement activity remains available for an

efficient opsonization of the persistent micro-organisms.

Complement and immunoglobulins in empyema 513Using a similar approach, we have also evaluated the concentrations and the integrity of

immunoglobulins in empyema fluids. Our results show that in the majority of culture-positivefluids, immunoglobulins of the IgG and IgM type are decreased when measured immunologically.It should be noted that with the method used, some culture-positive fluids had demonstrable levelsof IgG. Although it is likely that in some cases Fc fragments were quantitated as well as immunoglo-bulins during immunochemical measurements, there is also evidence for an incomplete breakdownof immunoglobulins in some empyemas.

Further evidence for an active breakdown of IgG in empyema was obtained by reconstitutionexperiments, which showed an inverse correlation between proteolytic activity and labelled IgGlevels in culture-positive effusions. Although this activity is probably favoured by the low level ofIgG substrate, the in vivo breakdown of opsonic factors occurs inappropriately in the presence of ahigh number of pathogenic organisms.

The mechanisms leading to complement and immunoglobulin catabolism in empyema areprobably multiple and remain conjectural: we have previously demonstrated the presence ofimmune complexes capable of complement activation, as measured with the C Iq-binding assay, inculture-positive fluids in contrast to bacteriologically sterile effusions (Perrin et al., 1975). Alternati-vely, incubation studies performed with bacteria isolated from empyemas have shown that thesemicro-organisms exhibited strong anticomplementary activity at concentrations detected in theseeffusions (Perrin et al., 1975). Finally, the role of lysosomal enzymes extruded from PMNLs has tobe considered: hydrolysis of IgG in abscesses by proteases of presumably leucocytic origin has beendocumented (Waller, 1974), and human PMNL granule extracts have been found to digest nativehuman IgG (Folds, Prince & Spitznagel, 1978). Activation of C3 (Wright & Gallin, 1977) as well asof other complement factors (Taylor, Grawford & Hugh, 1977) by human neutrophils has beenobserved under adequate experimental conditions. Our present findings are in accord with thesedata, and the demonstration of the reversal of in vitro proteolysis by the addition of normal serasuggests the possibility ofa relative or absolute localized deficit of protease inhibitors. However, ourstudy does not exclude the possibility either ofan additional intracellular proteolysis after ingestionby PMNLs, or of internal catabolism and excretion.

The presence of undetectable levels of available key functional complement components as wellas the breakdown of immunoglobulins in empyema fluid has major implications: immunoglobulinsand the complement system are essential in the defence against extracellular bacterial infections.Without these components, micro-organisms are not coated with antibody and opsonically effec-tive C3, so that PMNLs, even present in high numbers as is the case in empyema fluids, will only beable to ingest them at very low rates. Furthermore, other functions of antibody and complementsuch as neutralization of external bacterial components and bacteriolysis will fail to take place. Itcould be argued that our results merely reflect 'physiological' activation of complement in thepresence of bacteria: however, the fact that complement-dependent opsonic activity was almostundetectable in freshly obtained empyema fluid despite the presence of a large number of livemicro-organisms strongly suggests that the residual opsonic activity was unable to cope with thepersistent infection; breakdown ofimmunoglobulins in empyemas also argues in favour ofinappro-priate proteolysis of opsonic factors in this condition. Our results taken together suggest thatempyema fluid constitutes a localized, immune deficiency state, in which bacteria are protectedfrom the action of available serum components and immune reactants of the phagocytic system.

We are indebted to Thomas P. Stossel, Oncology Unit, Massachusetts General Hospital, Boston, for hiscriticisms and suggestions during the preparation of this manuscript. The secretarial help of Francoise Michaudis gratefully acknowledged.

Dr Daniel Lew is a recipient of a fellowship from the Swiss National Research Foundation (grant no83.460.1.76). This work was supported by the Swiss National Research Foundation (grant nos 3.847.077 and3.476.1.75), the Dubois-Ferriere-Dinu-Lipatti Foundation and the Frankfeld Foundation, Geneva.

5I4 D.P.Lewetal.

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