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SepsisRole of C5a in Multiorgan Failure During

Richard Erickson, John T. Curnutte and Peter A. WardGuo, Ellen M. Younkin, Robin G. Kunkel, Jiabing Ding,Stephanie R. McGuire, Vaishalee A. Padgaonkar, Ren-Feng Markus Huber-Lang, Vidya J. Sarma, Kristina T. Lu,

http://www.jimmunol.org/content/166/2/1193doi: 10.4049/jimmunol.166.2.1193

2001; 166:1193-1199; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Role of C5a in Multiorgan Failure During Sepsis1

Markus Huber-Lang,* Vidya J. Sarma,* Kristina T. Lu,* Stephanie R. McGuire,*Vaishalee A. Padgaonkar,* Ren-Feng Guo,* Ellen M. Younkin,* Robin G. Kunkel,*Jiabing Ding,‡ Richard Erickson,‡ John T. Curnutte,‡ and Peter A. Ward2*

In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well knowncomplication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model ofsepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOFoccurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressureof O2, rising partial pressure of CO2). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis withincreased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and tothe bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but bindingof fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatlyattenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotacticresponses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a preventsdevelopment of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects ofanti-C5a in the CLP model of sepsis. The Journal of Immunology,2001, 166: 1193–1199.

Sepsis in humans often leads to progressive multiorgan fail-ure (MOF),3 which is still associated with a high mortalityrate (1, 2). MOF in either humans or animals appears to

emerge as a consequence of progressive development of tissuehypoxia, complement activation, and an unregulated release intothe blood of a variety of proinflammatory mediators (ILs, cyto-kines, chemokines) (3–5). In humans with sepsis, there is wellestablished evidence for the appearance in plasma of complementactivation products, especially the anaphylatoxins (C3a, C4a, C5a)(6, 7). Persistent elevation of these anaphylatoxins appears to becorrelated with development of MOF and is inversely correlatedwith survival (7, 8). The complement activation product, C5a, is apotent neutrophil agonist that interacts with a seven-transmem-brane spanning receptor (C5aR) (9). Functional responses of neu-trophils, such as their release of proteases and generation of oxi-dants, as well as their enhanced adhesion to endothelial cells, maybe associated with development of direct or remote tissue injurythat occurs in sepsis and MOF (10). Neutrophils from patients withsepsis-induced MOF exhibit a loss of in vitro chemotactic respon-siveness to C5a (10), this functional deficit being correlated with aloss of the ability of C5a to bind to neutrophils (11). Furthermore,LPS- and C5a-induced neutropenia as well as neutrophil migration

into the peritoneum in models of acute endotoxemic shock can beinhibited by pretreatment with C5aR-antagonists (12, 13). Our re-cent studies of sepsis using the cecal ligation/puncture (CLP)model in rats have demonstrated the development of a substantialdefect in the ability of blood neutrophils to produce H2O2 whenstimulated in vitro with PMA (14). Neutrophil-generated H2O2 isa vital oxygen product required for myeloperoxidase-dependentintracellular killing of bacteria (15). The development of this de-fect in H2O2 production appears to be reversed by in vivo treat-ment of CLP animals with anti-C5a (14). Thus, there are severallines of evidence suggesting that sepsis triggers activation of com-plement and development of abnormalities in neutrophil function.These events may be associated with development of MOF.

In our original study of CLP-induced sepsis, the protective ef-fects of anti-C5a on survival were documented (14), but evidenceof MOF and the effects of anti-C5a on MOF were not assessed. Inthe current studies, we describe the progressive onset of MOF inthe CLP model and the multifunctional loss of chemotactic respon-siveness of blood neutrophils. We demonstrate the ability of anti-C5a to preserve chemotactic responsiveness of neutrophils and topreserve C5a binding sites on neutrophils, all of which are asso-ciated with greatly reduced development of MOF and lactate ac-idosis following CLP.

Materials and MethodsReagents

Unless otherwise specified, chemicals and reagents were purchased fromSigma (St. Louis, MO).

Preparation and characterization of Ab against rat C5a

Rat C5a peptide with the sequence KHRVPKKCCYDGARENKYET (cor-responding to amino acid residues 17–36) was coupled to keyhole limpethemocyanin and then used as an Ag to immunize rabbits. After severalimmunizations, the Ab was affinity purified from serum using the syntheticpeptide coupled to beads. The preparation of this Ab was performed byResearch Genetics (Huntsville, AL). In vitro, this polyclonal Ab immuno-precipitated from activated rat serum a protein aligning in Western blots

*Department of Pathology University of Michigan Medical School, Ann Arbor, MI48109; and‡Department of Immunology, Genentech, South San Francisco, CA 94080

Received for publication August 25, 2000. Accepted for publication October16, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by National Institutes of Health Research GrantHL-31963.2 Address correspondence and reprint requests to Dr. Peter A. Ward, Department ofPathology, University of Michigan Medical School, 1301 Catherine Road, Ann Ar-bor, MI 48109-0602. E-mail address: pward@umich.edu3 Abbreviations used in this paper: MOF, multiorgan failure; CLP, cecal ligation/puncture; rr, rat recombinant; GFR, glomerular filtration rate; ALT, alanine amino-transferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; LDH, lactatedehydrogenase.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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with the 14-kDa marker, consistent with the molecular mass of glycosy-lated C5a (data not shown). This Ab did not interfere with whole hemolyticactivity (CH50) in rat serum (data not shown).

Preparation of rat recombinant C5a (rr C5a)

A ‘his tag’ based on the TAGZyme system (Unizyme Laboratories, NewYork, NY) was engineered into the amino terminus of the rat C5a se-quence. The presence of the alternating histidine and glutamine residues (inall six, each alternating in tandem) facilitated purification of the recombi-nant C5a protein over a Ni21 Sepharose column. The his-tagged sequencewas cloned into the baculovirus vector pVL1393 (PharMingen, San Diego,CA) and expressed inSpodoptera frugiperda(Sf9) cells (PharMingen).Recombinant protein was then purified by lysing the cells in buffer con-taining 10 mM Tris pH 7.4, 130 mM NaC1, 1% Triton X-100, 10 mM NaF,10 mM NaH2PO4, 10 mM sodium pyrophosphate, and protease inhibitors(16 mg/ml benzamidine HCl, 10mg/ml aprotinin, 10mg/ml leupeptin, and10 mg/ml pepstatin). After removing the cell debris by centrifugation(10,0003 g for 10 min, then 100,0003 g for 1 h), the lysate was pouredover a Ni21 column (Qiagen, Chatsworth, CA). Then, rr C5a was elutedwith 10 mM Tris (pH 7.4), 300 mM NaCl, and 0–250 mM imidazole. Theeluate was dialyzed extensively with PBS (pH 7.4). This rr C5a was shownto be functionally active in a chemotaxis assay using rat blood neutrophils.

Experimental sepsis by CLP

Male Long-Evans specific pathogen-free rats (Harlan Breeders, Indianap-olis, IN) weighing 275–300 g were used in all experiments. Anesthesia wasinduced by i.p. administration of ketamine (20 mg/100 g body weight).After shaving the abdomen and applying a topical disinfectant, a 2-cmmidline incision was made, and the cecum was identified and ligated belowthe ileocecal valve, with care being taken not to occlude the bowel passage.The cecum was then subjected to a single “through and through” perfora-tion with a 21-gauge needle and was gently squeezed to ensure patency ofthe perforation sites. After repositioning the bowel, the abdominal incisionwas closed in layers with plain gut surgical suture 4-0 and skin clips (Ethi-con, Somerville, NJ). Sham-operated animals underwent the same proce-dure except for ligation and puncture of the cecum. Animals with CLPtreatment were injected i.v. immediately after induction of CLP with 400mg rabbit IgG against rat C5a (anti-C5a) or with 400mg preimmune rabbitIgG. Before and after surgery, animals had unrestricted access to food andwater.

For urine collection and determination of water intake and urinary out-put, rats were closely followed in metabolic boxes (Nalgene metaboliccage; Nalge Nunc International, Rochester, NY) from time 0–36 h after thesurgical procedures, as indicated in the individual experiments.

In some animals, a carotid artery catheter (PE-50; Becton Dickinson,Sparks, MD) was placed through an anterior cervical incision followed bys.c. tunneling and externalization in the posterior cervical area. Unlessotherwise indicated, arterial blood samples were collected at 12-h intervalsafter CLP to measure blood lactate, pH, pO2, pCO2, and HCO3

2 using ablood gas analyzer (OSM3, ABL 505, and EML 100; Radiometer, Copen-hagen NV, Denmark). In other groups of rats, blood was obtained from theinferior vena cava (at 12-h intervals up to 48 h after CLP) for biochemicalmeasurements and for isolation of neutrophils. Organs were processed formorphological studies as described below.

Transmission electron microscopy

Kidney tissues were sectioned into 1-mm3 cubes, fixed in 4% glutaralde-hyde in 0.1 M cacodylate buffer (pH 7.3) at room temperature overnight,and washed in two changes of 0.1 M cacodylate buffer (pH 7.3). Sampleswere postfixed in 2% OsO4 in 0.1 M cacodylate buffer (pH 7.3) for 1 h at25°C, dehydrated in graded series of alcohols for final dehydration in pro-pylene oxide, infiltrated with increasing mixtures of propylene oxide andEpon resin, and embedded in pure Epon. One-micron sections stained withtoluidine blue were evaluated by light microscopy. Thin sections wereobtained on an AO Ultracut ultramicrotome, stained with uranyl acetateand lead citrate, and evaluated in a Phillips 400 T electron microscope.

Measurement of biochemical parameters

Unless otherwise stated, blood was obtained from the inferior vena cava atthe time of sacrifice. Creatinine, blood urea nitrogen (BUN), alanine ami-notransferase (ALT), aspartate aminotransferase (AST), and lactate dehy-drogenase (LDH) were determined in serum samples by standard clinicalchemistry techniques. All assays were performed using commerciallyavailable reagents (Vitros dry film slides) on a Vitros 950 chemistry ana-lyzer (Ortho-Clinical Diagnostics, Rochester, NY). Electrolytes were de-termined by conventional flame spectrometry.

Calculation of glomerular filtration rates (GFRs)

The GFR was determined by endogenous creatinine clearance, measuredby the urine flow rate (ml/min) over a period of 36 h, and multiplied by theratio of the creatinine concentration in urine and in serum.

Urinary protein electrophoresis

After centrifugation of urine at 14,0003 g for 10 min, a 10-ml aliquot ofthe supernatant was added to 5ml of Laemmli’s sample buffer followed byboiling for 5 min. Samples were then electrophoresed on a 10% SDS-PAGE gel and stained with Coomassie blue.

Isolation of rat blood neutrophils

Whole blood from rats was drawn into syringes containing the anticoag-ulant ACD (Baxter Health Care, Mundelein, IL). Neutrophils were isolatedusing Ficoll-Paque gradient centrifugation (Pharmacia Biotech, Uppsala,Sweden) and dextran sedimentation. After hypotonic lysis of residual RBC,neutrophils were evaluated in chemotaxis assays and in binding studiesusing rr C5a and fMLP.

Chemotaxis assay

Following neutrophil isolation, cells were fluorescein labeled with BCECF(29,79-bis [2-carboxyethyl]-5-[and 6]-carboxy-fluorescein acetoxymethylester) (Molecular Probes, Eugene, OR). Labeled neutrophils (53 106 cells/ml) were then loaded into the upper chambers of 96-well minichambers(Neuroprobe, Cabin John, MD). Lower chambers were loaded with thechemotactic peptides C5a or fMLP. The upper and lower chambers wereseparated by a polycarbonate membrane of 3-mm porosity (Neuroprobe).Minichambers were incubated for 30 min at 37°C. The number of cellsmigrating through polycarbonate filters to the lower surface was measuredby cytofluorometry (Cytofluor II; PerSeptive Biosystems, Framingham,MA). Samples were measured in quadruplicate.

125I-rr C5a and [3H]fMLP binding studies

rr C5a (HQ]6C5a) was labeled with125I, using the chloramine T-basedmethod as described (16). This protocol allows gentle oxidation, whichpreserves the neutrophil chemotactic activity of C5a. Rat blood neutrophilswere obtained at 12, 24, 36, and 48 h after CLP and analyzed for the abilityof 125I-rr C5a to bind to these cells. For reference purposes, blood neutro-phils were also obtained from otherwise nonmanipulated healthy rats.Briefly, neutrophils (13 107 cells/ml) were incubated at 4°C in assaybuffer (HBSS, 0.1% BSA) in 1.5-ml microcentrifuge tubes with125I-rr C5a(specific activity, 35mCi/mg) in a final volume of 200ml. After incubationfor 20 min at 4°C, cell suspensions were layered over 20% sucrose andsedimented by centrifugation at 11,0003 g in a swing-out rotor for 2 min.After centrifugation, the tubes were frozen at280°C and the tips (con-taining the cell pellet) were cut off to determine the cell-bound125I-rr C5ausing a gamma counter (1261 Multig; Wallac, Gaithersburg, MD). Bindingaffinities were calculated in the conventional manner (17). Binding studieswith [3H]fMLP (0.1–100 nM) (specific activity, 96 Ci/mmol) (NEN, Bos-ton, MA) were performed similarly. Cell-associated [3H]fMLP was deter-mined in a Beckman LS 5000 TD liquid scintillation counter (Beckman,Palo Alto, CA). Specific binding was calculated by subtracting nonspecificbinding (in the presence of 100-fold excess of unlabeled fMLP; Sigma)from total binding.

Statistical analyses

All values are expressed as mean6 SEM. Data sets were analyzed with aone-way ANOVA. Individual group means were then compared using theTukey test. Significance was assigned wherep , 0.05.

ResultsReversal by anti-C5a of early respiratory alkalosis and latemetabolic acidosis in CLP animals

Arterial blood gases (expressed as mmHg) and pH values weredetermined in normal rats (ctrl) and in CLP rats that were infusedi.v. at time 0 (immediately after CLP) with either 400mg rabbitpreimmune IgG or 400mg anti-rat C5a IgG (anti-C5a). Blood sam-ples were obtained at early (24 h) and late time points (60 h) ofCLP. The results are shown in Table I. As expected, at 24 h therewas a mild respiratory alkalosis, with the arterial pH rising from7.47 6 0.01 to 7.536 0.01 in CLP rats treated with preimmuneIgG. In the same group, the blood pCO2 fell from a value of 38.66

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0.89 to 34.26 0.91. The HCO32 (mmol/L) and pO2 values were

unchanged in these two sets of animals at this early period of time.In contrast, in CLP rats treated with anti-C5a, the blood pH andpCO2 values at 24 h were the same as those values obtained innormal, unmanipulated rats.

When similar groups of rats were examined at 60 h, the CLPgroup treated with preimmune IgG showed evidence of metabolicacidosis, with the blood pH values falling from 7.486 0.003 to7.34 6 0.008, and blood HCO3

2 (mmol/L) falling from 26.660.47 to 20.86 1.86 (Table I). At the same time point, first signsof an additional respiratory acidosis with a mild hypercapnia werefound (increase in blood pCO2 from 36.66 1.29 to 44.56 3.32)and there was clear evidence of hypoxia, with a fall in the pO2

value from 1056 0.88 to 69.86 9.79. Remarkably, in the CLPgroup treated with anti-C5a, all values remained in the normalrange at 60 h (Table I).

Parameters of MOF and effects of anti-C5a treatment

The parameters of MOF are described in Table II as well as theprotective effects of anti-C5a. There was clear evidence of MOFdeveloping in CLP rats and the reversal of these parameters bytreatment of CLP rats with anti-C5a (400mg given i.v. at the timeof CLP) but not when preimmune IgG was used. Blood lactatelevels rose 2.64-fold during CLP. This rise was prevented in anti-C5a-treated rats. Evidence of liver dysfunction was indicated byelevations in serum bilirubin, ALT, and AST (2.5-, 5-, and 7-fold,respectively). In animals treated with anti-C5a, most of these pa-rameters remained in the normal range, whereas the rise in LDHlevels was attenuated. With regard to kidney function, in unpro-tected CLP animals, serum creatinine, BUN, and urine protein lev-els rose by 67, 280, and 150%, respectively, whereas in CLP ratstreated with anti-C5a, the levels were not statistically differentfrom those levels in sham animals. Electrophoretic analysis ofurine (SDS-PAGE, followed by Coomassie blue staining) revealedfaint amounts of albumin in urine from shams and greatly in-creased urinary albumin in CLP rats treated with preimmune IgG

involving lower and higher m.w. proteins relative to urinary albu-min. In CLP rats treated with anti-C5a, albumin was somewhatincreased above that found in sham rats, whereas the higher andlower m.w. bands were largely abolished (data not shown). Thesedata suggest defects both in glomerular filtration as well as intubular absorption. Urine output and GFR values in the unpro-tected CLP animals fell by 80 and 75%, respectively; thesechanges were greatly reduced in the animals receiving anti-C5a.Thus, manifestations of MOF in CLP animals are greatly attenu-ated in animals receiving anti-C5a.

Transmission electron microscopy of kidneys from CLP animals

Given the changes in proteinuria, urine output, and GFR (seeabove) we investigated presence of morphological changes in kid-neys by transmission electron microscopy. Tissue samples wereobtained 36 h after CLP-induced sepsis in rats treated with eitherpreimmune IgG or anti-C5a. As shown in Fig. 1, in CLP animalstreated with preimmune IgG, it was evident that there was exten-sive fusion of foot processes of podocytes in glomeruli (A, arrow-heads). In proximal convoluted tubules, the cells had lost their cellmembranes and exhibited mitochondrial swelling together with in-tracellular edema (B). In CLP animals that had been treated withanti-C5a, renal glomeruli appear normal with prominent podocytes(C), and epithelial cells of proximal convoluted tubules were intactand had normal structures (D).

Electrophoretic analysis of rr C5a

The relative purity of recombinantly produced rr C5a was deter-mined by electrophoretic analysis. Coomassie staining revealed asingle predominant band in the expected m.w. position for a non-glycosylated product (data not shown) This C5a preparation wasused both for functional (chemotaxis) assessment of rat blood neu-trophils and binding of125I-rr C5a to rat blood neutrophils.

Table I. Arterial blood gas analyses

Parameter

Early Phase (24 h) Late Phase (60 h)

ControlCLP 1

preimmune IgGCLP 1anti-C5a Control

CLP 1preimmune IgG

CLP 1anti-C5a

pH 7.476 0.01 7.536 0.01* 7.496 0.01 7.486 .003 7.346 .008* 7.496 .003pCO2 (mmHg) 38.66 0.89 34.26 0.91* 37.26 0.62 36.66 1.29 44.56 3.32 37.96 2.34HCO3

2 (mmol/L) 26.46 1.14 26.96 0.50 28.16 0.76 26.66 0.47 20.86 1.86* 26.46 1.14pO2 (mmHg) 1066 0.88 106.46 7.86 112.56 2.50 1056 0.88 69.86 9.79* 1066 4.17

p, Significant difference (p , 0.05) vs effects of anti-C5a treatment.

Table II. Effects of anti-C5a on parameters of MOF

Organ Parameter

Groups

Sham1 preimmune IgG CLP 1 preimmune IgG CLP 1 anti-C5a

Blood Lactate (nmol/L) 2.876 0.40 7.426 1.31 3.546 0.30Liver Bilirubin (mg/dl) 0.306 0.01 0.536 0.08 0.236 0.03

ALT (U/L) 46.66 1.8 255.06 65.2 63.66 2.4AST (U/L) 74.06 2.0 386.66 71.6 128.06 8.5LDH (U/L) 288.36 85.8 926.06 47.0 628.06 11.5

Kidney Creatinine (mg/dl) 0.436 0.03 0.706 0.05 0.436 0.06BUN (mg/dl) 11.66 1.4 44.56 12.2 17.66 0.7Urine protein (mg/dl) 1.306 0.04 3.276 0.29 1.576 0.19Urine output (ml/36 h) 26.26 0.8 6.66 1.0 19.06 1.0GFR (ml/min) 1.896 0.03 0.496 0.07 1.906 0.24

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Chemotactic dysfunction of blood neutrophils from CLP animals

The ability of blood neutrophils to respond in vitro to recombinantrat C5a and to the structurally unrelated chemotactic peptide,fMLP, both of which are known to be powerful chemotactic fac-tors for neutrophils, was assessed. Blood neutrophils were ob-tained from each of the three groups of rats (control and 36 h afterCLP from rats treated with either preimmune IgG or anti-C5a), asdescribed above. The isolated cells were labeled with a fluorescentprobe and evaluated by cytofluorometry for chemotactic responsesin vitro as described inMaterials and Methods. The results areshown in Fig. 2. When blood neutrophils were obtained from nor-mal rats (ctrl), chemotactic responses to rr C5a showed the ex-pected bell-shaped dose-response profile, starting at 0.1 nM andpeaking between 1 and 100 nM (Fig. 2A). Blood neutrophils fromCLP rats treated with preimmune IgG showed greatly diminishedchemotactic responsiveness over the entire range of concentrationsof rr C5a, whereas neutrophils obtained from CLP rats treated withanti-C5a showed responses that were not statistically differentfrom those of blood neutrophils obtained from normal rats.

The studies were extended to evaluate neutrophil responses tofMLP using similar protocols. The results are shown in Fig. 2B.Blood neutrophils from otherwise untreated rats showed the ex-pected pattern of dose response, progressing from 1029 M fMLPand reaching a plateau between 1026 and 1024 M, followed by adecline at high concentrations (1023 M) of fMLP. Blood neutro-

phils obtained from rats 36 h after CLP and treated with preim-mune IgG showed profoundly depressed chemotactic responsive-ness across the entire range of concentrations of fMLP (Fig. 2B,lowest line). In striking contrast, blood neutrophils obtained fromCLP rats that had been treated with anti-C5a had chemotactic re-sponses that were indistinguishable from those obtained fromblood of normal animals. Thus, blood neutrophils from CLP ratsacquired a chemotactic defect that was nonspecific, involving im-paired responses to both C5a and fMLP. In vivo blockade of C5ain CLP rats resulted in retention of normal chemotactic function inresponse to both C5a and fMLP.

Binding of125I- rr C5a to normal rat blood neutrophils

For binding studies, neutrophils were obtained as described above.As shown in Fig. 3A, over a range of 0.6–39.2 nM125I-rr C5a,there was progressively increased binding to neutrophils with aplateau in the binding occurring between 19.6 and 31.4 nM125I-rrC5a. When unlabeled rr C5a was mixed together with125I-rr C5ain a ratio of 100:1, the binding of the latter was almost totallysuppressed (Fig. 3A, lower line of graph). The computedKd valuewas 20 nM. For subsequent studies, the dose of 31.4 nM125I-rrC5a was used to determine whether there was defective bindingwhen blood neutrophils from CLP rats were used.

FIGURE 1. Transmission elec-tron micrographs of renal glomeruliand proximal tubular epithelium36 h after onset of CLP in animalstreated (at time 0) with 400mg pre-immune IgG (Aand B) or anti-C5a(anti-C5a) (Cand D). In A, arrow-heads indicate extensive fusion offoot processes of podocytes in glo-meruli, which did not occur in anti-C5a treated animals (C). Proximaltubular epithelial cells have intactnuclei but show intracellular edema,loss of the cell membrane, and mi-tochondrial swelling (B), features ofwhich were not seen in anti-C5a-treated animals (D). A andC, 34600;B andD, 33500.

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Defective binding of rat C5a to blood neutrophils from animalswith CLP

These studies were undertaken to assess the status of binding of125I-rr C5a to blood neutrophils as a function of time after onset ofCLP. Neutrophils were isolated from blood at 0, 12, 24, 36, and48 h after onset of CLP in animals that had been treated i.v. at time0 with 400 mg preimmune IgG or anti-C5a. The results of thesebinding studies are shown in Fig. 3B. Binding values of;30,000cpm with a nonspecific binding fraction of;5000 cpm (Fig. 3B)were found in blood neutrophils obtained from normal rats (time0). When CLP rats were pretreated with 400mg preimmune IgGand blood neutrophils were harvested at 12, 24, 36, and 48 h afterCLP, the specific binding values for rr C5a relative to the 0 timevalues had fallen to 3.96, 4.35, 33.2, and 40.25%, respectively. InCLP animals that had been infused with anti-C5a, the results for12, 24, 36, and 48 h were significantly different from that of pre-immune IgG-treated rats. Specific C5a binding values at these timepoints were 40.7, 61.2, 76.7, and 92.3%, respectively. Thus, itappears that in CLP rats, treatment with anti-C5a caused retentionof the binding sites in blood neutrophils. Whether this was due tooccupancy by C5a of C5aR or due to internalized C5a-C5aR com-plexes remains to be determined.

Binding of [3H]fMLP to rat blood neutrophils

Binding of [3H]fMLP to rat neutrophils isolated from normal ratblood was determined as described above. As seen in Fig. 4, there

was a progressive increase in binding over a range of 0.1–100 nMfMLP, reaching a plateau at a concentration of 50 nM or higher(Kd 5 35 nM). Nonspecific binding was determined by incubatingneutrophils with a 100:1 ratio of nonlabeled fMLP to [3H]fMLP.Neutrophils obtained from normal rats or from CLP rats (24 h afterCLP) treated with either 400mg preimmune IgG or 400mg anti-C5a did not show statistically significant differences in binding of

FIGURE 4. Binding of [3H]fMLP to normal rat neutrophils (F) with acalculatedKd value of 35 nM and binding to neutrophils obtained 24 h afterCLP from rats injected with 400mg preimmune IgG (f) or anti-rat C5a(anti-C5a) (Œ). Nonspecific binding was determined by binding in the pres-ence of 100-fold excess of cold fMLP (E).

FIGURE 2. Chemotactic responses of blood neutrophils obtained fromnormal rats (ctrl) or from CLP rats (at 36 h) treated with 400mg preim-mune IgG or 400mg anti-rat C5a (anti-C5a) at time 0. In vitro chemotacticresponses to rr C5a (A) and fMLP (B) were measured by cytofluorometry.For each data point,n 5 4. Asterisks represent statistical differences (p,0.05) between neutrophil responses obtained in cells from CLP animalstreated with anti-C5a and CLP animals treated with preimmune IgG.

FIGURE 3. Binding of 125I-rr C5a to neutrophils obtained from normalrat blood (A). Details are described inMaterials and Methods. The calcu-latedKd value was 20 nM. The ability of unlabeled rr C5a (in a ratio of100:1) to compete with binding of125I-rr C5a is also shown. InB, bloodneutrophils were obtained at the times indicated (after onset of CLP), andbinding of 125I-rr C5a was determined. Neutrophils were obtained fromCLP rats treated with 400mg preimmune IgG or anti-rat C5a (anti-C5a).

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[3H]fMLP (50 nM). Thus, CLP does not reduce fMLP binding inblood neutrophils, in striking contrast to the situation with C5abinding (Fig. 3), even though the chemotactic responses of fMLPare greatly suppressed (Fig. 2B).

DiscussionThe role of complement activation products in sepsis and in com-plications such as MOF is a debatable issue. There is fairly goodevidence to suggest that, in the absence of complement such as incomplement-depleted animals (14) or in C3 or C4 mutant micelacking this critical complement component (18, 19), animals arehighly susceptible to the early and lethal effects of experimentalsepsis. These observations reinforce the long-standing concept thatthe complement system functions as a critical protective pathwayvia products such as C3b and iC3b. Evidence also exists that, dur-ing sepsis, extensive activation of the complement system occursresulting in loss of homeostasis, which may in some manner com-promise survival. Intravenous infusion of C5a into dogs generateda shock syndrome characterized by portal blood pooling and anassociated decrease in venous return, cardiac output, and arterialpressure (20). In experimental sepsis caused by massive i.v. infu-sion of live, Gram-negative bacteria into pigs or monkeys, devel-opment of biochemical and hemodynamic abnormalities, and earlydeath have been described within 4–6 h. Some of these eventswere partly attenuated if the pigs were first treated with a mAb tothe carboxyl-terminal region of porcine C5a or monkeys treatedwith a polyclonal Ab to whole human C5a (21–23). Our own stud-ies have indicated that lethality associated with CLP-induced sep-sis in rats can be significantly diminished by treatment of animalswith anti-C5a (14). This treatment significantly preserved the re-spiratory burst of blood neutrophils, thus maintaining the H2O2

response (14), which is vital for the effective myeloperoxidase-dependent pathway of bactericidal activity of neutrophils (15).Collectively, these studies suggest that sepsis may be associatedwith excessive activation of the complement system, resulting indefects in bactericidal function of neutrophils.

In this study, CLP in rats caused a time-dependent pattern ofMOF, with a late onset of hypoxia (60 h) in CLP rats treated withpreimmune IgG but not in CLP rats after blockade of C5a. In ashort-term sepsis model (4–6 h), pigs or monkeys challenged withvery high doses of liveEscherichia coliand treated with an Ab toporcine or human C5a showed (in comparison to otherwise un-treated animals with sepsis) a partial improvement in some func-tional parameters (such as oxygenation, lung edema, and oxygenuptake) and an improvement in the oxygen extraction ratio accom-panied by an attenuated increase in lactate levels (21–23). Hyper-lactatemia is known to be a reliable indicator of advancing MOF inhumans (24). In CLP animals treated with preimmune IgG, bloodlactate levels were very high at 48 h. This elevation was abrogatedin CLP rats treated with anti-C5a. Data in this report clearly indi-cate that CLP animals develop evidence of progressive lung, kid-ney, and liver failure that is C5a dependent. All of these outcomesmay be linked to excessive C5a generation during sepsis, resultingin a compromised respiratory burst of neutrophils and their oxy-gen-dependent bactericidal pathways.

Our data also document in the CLP model in rats that bloodneutrophils acquire a significant defect in chemotactic responsive-ness both to C5a as well as to fMLP. Because these two chemo-tactic peptides are structurally totally distinct and use different re-ceptors on neutrophils (25, 26), our observations may imply apostchemotactic factor receptor-acquired functional defect. Thismay be the result of excessive in vivo stimulation by C5a, resultingin defects in signal transduction that extend to agonists beyondC5a and impair chemotactic responsiveness to C5a as well as to

other chemotactic factors. Our studies document development ofchemotactic defects in blood neutrophils from CLP rats involvingresponses to both C5a and to fMLP. These defects are associatedwith loss of binding of rat125I-rr C5a to neutrophils but normalbinding of [3H]fMLP. fMLP is known to be the major neutrophilchemotactic factor in culture filtrates ofE. coli (27). These datawould support the possibility that in vivo contact of neutrophilswith C5a induces a downstream defect in signal transduction be-yond a point at which C5aR and fMLP-receptor (fMLPR) path-ways merge. In other words, although blood neutrophils from CLPrats appear to demonstrate normal binding of fMLP, this binding isunable to effectively engage a post receptor signaling pathway thatis common to both C5aR and fMLPR. This is consistent with ear-lier studies in which, when neutrophils were exposed to low levelsof C5a (present in activated serum), the cells acquired a chemo-tactic defect specific to C5a. When neutrophils were exposed tohigher concentrations of C5a, they then acquired a broad defect inchemotactic function characterized by loss of responsiveness toC5a and to bacterial chemotactic peptides (11). In vivo, the che-motactic responses of neutrophils obtained from patients with sep-sis or multisystem trauma with adult respiratory distress syndromewere found to be specifically deactivated to C5a or deactivated toboth C5a and fMLP depending on clinical severity and time aftertrauma or onset of sepsis. In contrast, no alteration in neutrophilfunction was found in patients who did not develop adult respira-tory distress syndrome (28). Our recent studies in the CLP modelindicate that the H2O2 response of neutrophils to PMA is impaired(14), suggesting a broad defect in postreceptor signaling pathways.

Perhaps relevant to the chemotactic dysfunction of blood neu-trophils described in this report is the finding that binding sites forrat C5a (but not for fMLP) on neutrophils have been virtually lost(,5% of binding in control neutrophils) during the first 24 h ofsepsis. By 48 h, neutrophils still demonstrated,50% of C5a bind-ing, in striking contrast to nearly normal binding values of C5a forneutrophils obtained from CLP rats treated with anti-C5a (Fig. 3).Under these latter conditions, chemotactic responsiveness to C5aand to fMLP was totally preserved. These data are in accord withfindings of specific loss of C5a binding (but intact fMLP binding)to neutrophils in patients with sepsis (11). It seems possible thatthe mechanism by which anti-C5a may protect against develop-ment of MOF during sepsis is its ability to cause preservation inthe chemotactic and respiratory burst function of blood neutrophilsby intercepting C5a before it has a chance to bind to C5aR onneutrophils. Retention of these functional responses would facili-tate effective in vivo recruitment of neutrophils and maintenance ofbactericidal function in these cells. Understanding the relationshipbetween C5a and the induction of host defense dysfunction inblood neutrophils is the topic of current focus.

AcknowledgmentsWe thank Beverly Schumann and Peggy Otto for excellent secretarial as-sistance. We also thank Faith Bjork and Dr. D. McConnell for iodinationof C5a. In addition, we thank Dr. D. Giacherio for assistance in biochem-ical measurement of blood samples.

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