1987 70: 1743-1749   

 F Dallegri, A Ballestrero, G Frumento and F Patrone vitro from human monocytesAugmentation of neutrophil-mediated erythrocyte lysis by cells derived in

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Blood, Vol 70. No 6 (December), 1987: pp 1743-1749 1743

Augmentation of Neutrophil-Mediated Erythrocyte Lysis by Cells Derived

In Vitro From Human Monocytes

By Franco Dallegri, Alberto Ballestrero, Guido Frumento, and Franco Patrone

Neutrophilic polymorphonuclear leukocytes (PMNs) wereincubated with opsonized zymosan and lysed human eryth-

rocytes (RBCs) as measured by a 51Cr release method.Conversely. myeloperoxidase (MPO)-negative hydrogenperoxide (H202)-generating cells. derived in vitro fromhuman monocytes (monocyte-derived cells (MDCs). wereineffective per se but capable of augmenting the lysis byPMNs. The lysis by PMNs and PMNs plus MDCs wasinhibited by catalase. azide. taurine. and alanine. consistent

with the requirement for hypochlorous acid (HOCI). Asdetected under conditions similar to those used for lyticassays. MDCs failed to produce HOCI but augmented theHOCI recovery from the PMN-RBC system. Moreover.when the extent of the lysis was plotted as a function ofthe HOCI recovery. a positive linear relationship was found.

Although the actual size of the H202 extracellular pool

W HEN TRIGGERED by appropriate stimuli, either

soluble (phorbol myristate acetate) or particulate

(opsonized zymosan [OPZ], heat-aggregated IgG), neutro-

phils (neutrophilic polymorphonuclear leukocytes [PMNs])

have been shown to be capable of lysing a wide spectrum of

eukaryotic target cells.’�5 These in vitro systems of cytolysis

have been used to understand the mechanisms by which

PMNs may injure tissue cells at inflammatory sites. In the

present paper, by the use of a previously characterized

system constituted of OPZ-triggered PMNs and human

erythrocytes (RBCs) as targets,6 we provide evidence that

the PMN cytolytic efficiency can be augmented by myelo-

peroxidase (MPO)-negative cells derived in vitro from

human monocytes (MDCs) and per se incapable of mediat-

ing lysis. The results uncover a novel possible role for

MPO-negative mononuclear phagocytes in interaction with

PMNs during inflammatory processes.

MATERIALS AND METHODS

Preparation of PMNs. Heparinized venous blood (heparin, 10

U/mL, Liquemin, Roche, Milan, Italy) was obtained from healthymale volunteers. PMNs were isolated by dextran sedimentation andsubsequent centrifugation on a Ficoll-Hypaque density gradient as

previously described.’ Contaminating erythrocytes were removed byhypotonic lysis.6 The resulting PMNs were washed three times with

Hanks’ balanced salt solution lacking phenol red (HBSS, FlowLaboratories, Ltd, Irvine, Scotland) and resuspended in HBSS

supplemented with 0.1 mg/mL bovine serum albumin (BSA, Cal-

biochem-Behring, San Diego) (BSA-HBSS). Final cell suspensionscontained 97% or more PMNs and more than 98% viable cells asevaluated by the ethidium bromide-fluorescein diacetate test.7

Preparation of MDCs. Venous blood (350 mL) from healthymale volunteers was collected into 53 mL of anticoagulant citrate-phosphate-dextrose-adenine (CDPA) (Baxter, Trieste, Italy). Thebuffy coat, obtained two to three hours after venipuncture, wasdiluted with an equal volume of 0.9% NaCI, and 25 mL of the cellsuspension was layered on 15 mL Ficoll-Hypaque (Nyegaard & Co,

Oslo) in 50-mL polypropylene conical tubes (No. 25330, CorningGlass Works, Corning, NY). After centrifugation (400 g, 30 mm-

utes, 22#{176}C),the upper cell layer (mononuclear cells) was collectedand spun down at 400 g for ten minutes at 22#{176}C.The sedimented

cells were washed three times with HBSS (200 g, ten minutes, 22#{176}C)

could not be measured because of the inexistence of areliable assay to probe our cytolytic model without per-turbing the equilibrium of the system. the results pre-

sented suggest that MDCs enhance the PMN-mediatedlysis by improving the HOCI production. presumably bysupplying extra amounts of H302 to be handled by PMNMPO. In fact. the events mediated by MDCs could bereproduced by using an appropriate H202-generating enzy-matic system (glucose-glucose oxidase). The present

study provides direct evidence for the possibility of cooper-ation between MPO-positive and MPO-negative phago-cytes in exerting functions (HOCI production and. in turn.cytolysis) possibly relevant to the outcome of inflammatoryprocesses.a 1987 by Grune & Stratton, Inc.

and resuspended in Fisher’s medium (Flow) supplemented with 25%filtered horse serum (HS, Flow), 2 mmol/L glutamine, 100 U/mLpenicillin, and 100 ,Lg/mL streptomycin (US-Fisher’s medium8).

The cells were then plated in 75-cm2 tissue culture flasks (Sterilin,Ltd. Teddington, England) in thin suspension layers (2 x l07/mL, 5

mL). After incubation for two hours at 37#{176}Cin a humidifiedatmosphere of 95% air and 5% C02, nonadherent cells were removed

by washing five times with prewarmed (37#{176}C)HBSS and theadherent cells overlaid with 8 mL HS-Fisher’s medium. The flaskswere then returned to the CO2 incubator. Three days later, the

medium containing detached cells was decanted, and adherent cells

were rinsed three times with prewarmed (37#{176}C)HBSS. Adherentcells were then removed by pipetting after incubation (20 minutes,

4#{176}C)with phosphate-buffered saline (PBS, pH 7.4) containing 0.2%

EDTA (Sigma Chemical Co. St. Louis) and 5% heat-inactivated (30minutes, 56#{176}C)fetal calf serum (Flow).9 The resulting cells were

centrifuged (200 g, ten minutes, 4#{176}C),washed three times with cold

(4#{176}C)HBSS, and resuspended in cold (4#{176}C)BSA-HBSS. Only cellsuspensions containing >90% a-naphthyl acetate esterase-positivecells, as determined by the method of Li et al,’#{176}were used. Thesecells, referred to as MDCs, were >97% viable as judged by theethidium bromide-fluorescein diacetate test.7

Preparation of OPZ. OPZ was prepared by incubating a sus-pension containing 10 mg/mL boiled zymosan (Sigma) in PBS (pH7.4) with an equal volume of fresh human serum for 30 minutes at

37#{176}C.The particles were then washed three times with PBS andresuspended in BSA-HBSS at the appropriate concentration.

Measurement of RBC lysis. The RBC lysis was studied by a

From the First Medical Clinic, University of Genoa Medical

School, Italy.

Submitted February 17. 1987; accepted July 29. 1987.

Supported by the Italian Consiglio Nazionale delle RicercheFinalized Project “Oncology” No. 86.00518.44/115.11547.

Address reprint requests to Franco Dallegri. MD, ISMI-Clinica

Medica I, Universita’ degli Studi, Viale Benedetto XV, n. 6. 1-16132Genova, Italy.

The publication costs ofthis article were defrayed in part by page

charge payment. This article must therefore be hereby marked“advertisement” in accordance with /8 U.S.C. §1 734 solely to

indicate this fact.© I 987 by Grune & Stratton, Inc.

0006-4971/87/7006-0006$3.00/0

For personal use only. by guest on July 10, 2011. bloodjournal.hematologylibrary.orgFrom

1744 DALLEGRI ET AL

�‘Cr release method as previously described.6 Briefly, RBCs were

washed four times with HBSS and labeled (60 minutes, 37#{176}C)with100 to 200 MCi Na2[5’Cr104 (Radiochemical Centre, Amersham,

England). The cells were then washed three times with HBSS and

resuspended in BSA-HBSS. When required, RBCs were pretreated

(60 minutes, 37#{176}C)with 20 mmol/L 3-amino-l,2,4-triazole (AT,Sigma) plus 2 mmol/L L-ascorbic acid (Sigma) (RBCAT) to inhibit

catalase activity.” A quantity of l0� RBCs were mixed with PMNs,

MDCs, or PMNs plus MDCs at appropriate concentrations. In someexperiments, MDCs were replaced by glucose oxidase (GO, type II,Lot I I 1F-9605, Sigma) dissolved in HBSS (prepared without glu-cose) and mixed with the other components of the reaction to reach afinal concentration of 5 mU/mL (incubation medium, BSA-HBSSwith 1 mg/mL glucose). Incubations were initiated by the addition

of OPZ (0.4 mg/mL) and followed by centrifugation of the samples

at 50 g (five minutes, 22#{176}C).Tests were performed in triplicate and

in a final volume of 200 ILL in round-bottomed microplates (Steril-

in). After incubation for four hours at 37#{176}Cin a humidified

atmosphere of 95% air and 5% CO2. the �‘Cr release was determined

in the cell-free supernatants. The percentage of cytolysis (percent

5’Cr release) was calculated according to the formula (E - S)/(T - 5) x 100, where E is the counts per minute released in the

presence of effector cells, T is the counts per minute released after

lysing target cells with 5% Triton X-lOO (Sigma), S is the counts per

minute spontaneously released by target cells in the absence of

effector cells (in each case, <10%).

Measurement of H202 release. The hydrogen peroxide (H2O2)release was studied by a modification of the homovanillic acid(HVA)-horseradish peroxidase (HRP) method’2 as previouslydescribed.” Tests were performed in triplicate under experimental

conditions similar to those used for cytolytic assays except that

incubations were done in Falcon plastic tubes ( I 2 x 75 mm, Falcon

Labware, Oxnard, CA). Briefly, 5 x iO� PMNs were incubated

(37#{176}C)with and without 0.4 mg/mL OPZ in the presence of 200

Mmol/L HVA (Sigma) plus 2 U/mL HRP (Sigma). When required,2 x 10’ RBC targets were added. Similar experiments were carried

out with an appropriate number of MDCs both in presence and inabsence of PMNs. The cells were sedimented by centrifugation (50g, five minutes) at the beginning of the assay. The reactions,

performed in a final volume of 2 mL, were stopped after 20 minutes

with 0.25 mL of0.l mol/L glycine-NaOH buffer (pH 12) contain-

ing 25 mmol/L EDTA. The amount of H202 in the cell-free

supernatants was determined fluorimetrically (X,�, = 3 1 2 nm,

Acm 420 nm) by using appropriate standards of H2O2. Preliminary

experiments showed that the amount of H202 recovered fromOPZ-triggered neutrophils incubated (20 minutes) as a pellet equalsthat recovered from OPZ-triggered neutrophils incubated as a pellet

during the first five minutes of the assay period (20 minutes) and

then resuspended and maintained in suspension.

Measurement of HOCI production. The generation of hypo-chlorous acid (HOCI) by OPZ-triggered PMNs was quantitated bythe taurine trapping technique’3 as previously described.” Theconditions of the assay were identical to those used for the H202

release except that the reactions were carried out in a final volume ofI mL. Briefly, an appropriate number of PMNs was incubated (20

minutes, 37#{176}C)with 0.4 mg/mL OPZ in the presence of 20 mmol/Ltaurine (Sigma). Tests were performed in the presence and in theabsence of 2 x 106 RBC targets or 300 units catalase (bovine liver,

Sigma; amount of enzyme chosen on the basis of preliminary

experiments). Similar experiments were carried out with MDCsboth in presence and in absence of PMNs. The cells were sedimentedby centrifugation (50 g, five minutes) at the beginning of the assay.At the end of the incubation period (20 minutes), catalase (600

units, Sigma) was added to reduce any residual H2O2. The sampleswere then incubated (22#{176}C,five minutes) with 100 nmol of5-thio-2-nitrobenzoic acid (Nbs) prepared by reducing 5-5’-dithiobis

(2-nitrobenzoic acid) (Sigma) as described by Aune and Thomas.’4The amounts of Nbs oxidized were determined by measuring theabsorbance at 412 nm of the cell-free supernatants (� = 1.36 x i04mol/L/cm). The quantities of HOCI trapped by taurine (yelding

taurine chloramine) were calculated by taking into account that 1mol taurine chloramine oxidizes 2 mol Nbs.’3 As for the H202 assay,the method of HOC1 detection maintained its efficiency also when

tests were carried out with cells incubated as a pellet for 20 minutes.

Under the conditions described earlier, the uptake of taurine chlo-ramine by RBCs, via the 4,4’-diisothiocyano-2,2’-disulfonic acid

stilbene (DIDS)-inhibitable anion transport system,’5 did not affectthe taurine chloramine recovery (the amounts of taurine chloraminerecovered in the presence of RBCs equalled those recovered in the

presence of RBCs pulsed (30 minutes) with 0.1 mmol/L DIDS(Sigma).”

Enzyme assays. The total cell content of MPO was measuredafter lysing the cells by sonication followed by freezing-thawing andsolubilization with 0.5% hexadecyltrimethylammonium bromide

(HTAB, Sigma).’6 The MPO activity released by cells incubated

(one hour, 37#{176}C)with 0.4 mg/mL OPZ was determined in HTAB-untreated cell-free supernatants. The MPO assay was done by an

O-dianisidine method as previously described.” One unit of enzyme

activity was defined as that oxidizing I izmol of O-dianisidine/min at

25#{176}C(optical density, 550, t - 1 1.3 mmol/L/cm).

For determination of RBC catalase activity, the cells were lysed asdescribed by Beutler,’7 and the enzyme activity was determined by

recording the rate of decomposition of H202 at 230 nm (t - 0.071mmol/L/cm).’7 The activity of “reagent” catalase (bovine liver,Sigma) was measured similarly. The results were expressed asnanomoles H202/min at 25#{176}Cor units (1 unit of enzyme activity was

defined as that catalyzing the breakdown of I �mol H202/min at

25#{176}C).Special materials. Superoxide dismutase (SOD, type 1, bovine

blood, Sigma), stored at - 20#{176}Cat a concentration of 5 mg/mL in

distilled water, was diluted in medium before use. Catalase (bovine

liver, 40,000 U/mg protein), taurine, L-alanine, mannitol, andbenzoate were purchased from Sigma. Sodium azide (NaN3) and

(NH4)2S04 were from Merck, Darmstadt, FRG. MPO, as a super-natant from the PMN granule fraction (MPO 4,800 mU/mL, 24.9mg/mL protein), was prepared by using normal human PMNs andstored at - 20#{176}C.’8Other reagent-grade compounds were used as

obtained from commercial suppliers. In some experiments, BSA-

HBSS medium was replaced with a Cl-free, 0.1 mol/L phosphate

buffer (pH 7.4) with I .5 mmol/L MgSO4 supplemented with Img/mL glucose (Merck) and 0.1 mg/mL BSA (Calbiochem-

Behring)(Cl-free medium).

RESULTS

As depicted in Fig 1 , OPZ-triggered PMNs lysed RBC

targets, whereas MDCs were ineffective. When added to the

PMN-RBC system (0.25 x l0� PMNs plus l0� RBCs,

PMN:RBC ratio, I :4), MDCs enhanced the lysis in a

dose-dependent manner (Fig 2). As detailed in Table 1, the

lysis in the complete PMN-MDC-RBC system (0.25 x l0�

PMNs plus 0.25 x l0� MDCs plus I0� RBCs) was inhibited

by adding catalase to destroy H202 (heat-inactivated cata-

lase was ineffective), azide to inhibit MPO, and amino acids

(alanine, taurine) to scavenge HOCI. Scavengers of superox-

ide anion (SOD) and hydroxyl radicals (mannitol, benzoate)

were ineffective (Table 1). Similar results were obtained in

the PMN-RBC system (Table I), in agreement with our

previous findings.6” Moreover, the absence of Cl corn-

pletely prevented lysis in the PMN-MDC-RBC system (per-

cent 51Cr release, 0. 1 ± 0.8 and 36.0 ± 2.3 in the absence and

For personal use only. by guest on July 10, 2011. bloodjournal.hematologylibrary.orgFrom

‘450

40

30

20

.�.10

C-,

�! � �

40 I-

30

20

10

01

MDCs

0.031 0.062 0.125 0.25

added tiio��j

Fig 2. Augmentation of the OPZ-triggered PMN-mediatedRBC Iysis by MDCs. The abscissa shows the number of MDCsadded to the PMN-RBC system (0.25 x 10’ PMNs plus i0RBCs/weIl). Each point represents i ± 1 SD of at least fourexperiments.

PMN5, 5 x 10’; MDCs, 5 x io�; RBCs, 2 x 106.

tIn nmol/2O mm, i ± 1 SD of three experiments.

MDCS ENHANCE RBC LYSIS BY PMNs 1745

0.125 0.25 0.5 1

PMNs (.1 and MOCs .1 , [x1O�)

Fig 1 . OPZ-triggered RBC lysis by PMNs or MDCs. The num-ber of RBCs is 1 08/weIl. Each point represents i ± 1 SD of at leastthree experiments.

in the presence of Cl respectively, ic ± 1 SD, n = 3). Simi-

larly, Cl was required for RBC lysis by PMNs.’9 Finally,

the addition of 20 rnmol/L NH4� caused >95% inhibition of

the lysis in both the PMN-RBC and the PMN-MDC-RBC

systems. These data suggest that amplification of PMN-

mediated lysis by MDCs requires HOC1 production via the

MPO-H2O2-C1 system.

To gain further insight into the mechanism whereby

MDCs enhance PMN-mediated lysis, we measured the

recovery of H2O2 and HOCI generated by PMNs and MDCs

Table 1 . Effect of Scavengers and Inhibitors of Oxygen-ReactiveSpecies on the OPZ-Triggered RBC Lysis by

PMNs Plus MDCs or by PMNs

Percent RBC Lysis by

Additive PMNs + MDCs PMNs

None 35.6 ± 2.5 49.3 ± 5.0

SOD, 300 U/mL 35.6 ± 4.0 47.3 ± 2.3

Catalase,4,000U/mL 0.1 ± 1.1 -0.1 ±0.7

Azide, 1 mmol/L 5.6 ± 2.7 8.0 ± 4.2

Taurine, 20 mmol/L 4.6 ± 1.8 5.9 ± 2.5

Alanine, 20 mmol/L 4.5 ± 2.5 4.5 ± 3.1

Mannitol, 20 mmol/L 35. 1 ± 4.2 47.0 ± 2.6

Benzoate, 20 mmol/L 34.8 ± 3.7 50.9 ± 7.4

PMN-MDC-RBC system, 0.25 x i0� PMNs plus 0.25 x 10� MDCs

plus i05 RBCs; PMN-RBC system, 10� PMNs plus � RBCs. Results are

expressed as i + 1 SD of three experiments.

under conditions similar to those used for cytolytic assays

(cells incubated as a pellet). As shown in Table 2, both

PMNs and MDCs, incubated with OPZ in absence of RBCs,

released H2O2. Nevertheless, only PMNs produced HOCI

(Table 2), consistent with the absence of detectable MPO

activity in MDCs (MDC MPO content, undetectable; PMN

MPO content, 157.2 ± 1 1.3 mU/106 PMNs, OPZ-triggered

MPO release, 4.2 ± 0.6 mU/l06 PMNs/lh, � ± I SD,

n = 4). When MDCs were added to PMNs in absence of

RBCs, the H202 recovery increased, but the HOCI recovery

did not change (Table 2). It seems therefore that the HOCI

production cannot be further enhanced by the extra amount

of H202 supplied by MDCs. This is in agreement with the

incapacity of the H202-generating enzymatic system, glu-

cose-GO, to increase the HOC1 production by OPZ-

triggered PMNs (HOCI recovery, 1 5.5 ± 2.6 and 1 5.9 ± 2.6

nmol/5 x l0� PMNs/20 mm from PMNs incubated alone

or with 5 mU/mL GO respectively; 5 mU/mL generated GO

2.3 ± 0.5 nmol/H202/min, � ± 1 SD, n = 3). In ancillary

experiments carried out with cells in suspension instead of

centrifuged as a pellet, the HOC1 production by OPZ-

triggered PMNs could not be augmented by adding 5

rnU/mL GO or 16 mU/mL MPO from the supernatant of

the PMN granule fraction (92.8 ± 6.9, 92.8 ± 7.3, and

91.5 ± 9.2 nmol HOC1/106 PMNs/l h from PMNs incu-

bated alone, with GO, and with MPO respectively, � ± I SD,

n=3).

When RBCs were added to PMNs to complete the system

(PMN:RBC ratio, 1:4; number of RBCs 2 x 106; cells were

incubated as a pellet) as in the cytolytic assays, a decreased

Table 2. Recovery of H202 and HOCI From OPZ-Triggered PMNs

and/or MDCs in the Presence and in the Absence of RBCs

Assay Conditions

Extracellular Recovery�

H202 HOCI

PMNs 15.3 ± 2.7 14.8 ± 1.2

MDCs 7.4±1.0 0

PMNs + MDCs 21.3 ± 3.5 14.1 ± 1.4

PMNs + RBCs 7.6 ± 1.3 8.1 ± 0.9

MDCs + RBCs 4.6 ± 1.7 0

PMNs + MDCs + RBCs 12.3 ± 1.4 12.8 ± 1.2

For personal use only. by guest on July 10, 2011. bloodjournal.hematologylibrary.orgFrom

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6 8 10

HOCI recovery I I) mel

12 14 16

20

�1oC.,

r’ir’iA B C 0

1746 DALLEGRI ET AL

H2O2 and HOCI recovery (-50%) was found (Table 2). This

reduction in HOC1 generation results in a relative impair-

ment of the HOCI-dependent PMN lytic efficiency.6 As

summarized in Table 2, MDCs increased the H2O2 and

HOC1 recovery from the PMN-RBC system. Moreover,

when the extent ofthe lysis by PMNs and PMNs plus MDCs

(Figs 1 and 2) was plotted as a function of the HOCI

recovery detected under conditions similar to those used for

cytolysis (identical cell ratios but with 2 x 106 RBCs instead

of l0� RBCs used in lytic assays), a positive linear relation-

ship was found (Fig 3). Together, these results suggest the

possibility that MDCs amplify the lysis primarily by improv-

ing the HOCI production in the PMN-RBC system, possibly

through an effective restoration of the PMN H202 extracel-

lular pool depleted by RBC targets. Three additional find-

ings are consistent with this possibility. First, the replace-

ment of RBC targets with RBCAT (RBCAI had a >94%

reduction in catalase activity; catalase activity of native

RBCs 4,258 ± I 12 nmol H2O2/min/lO6cells, � ± 1 SD,

n = 6), incapable of efficiently consuming PMN-derived

H202,6 completely abrogated the MDC capacity to augment

the PMN-mediated HOCI-dependent (not shown) lysis (per-

cent increment, 1.1 ± 3.7, i ± 1 SD, n = 3) and the HOC1

recovery from the system (percent increment, -0.02 ± 8.2,

� ± I SD, n = 4). Second, enhancement of the HOCI pro-

duction by MDCs in the PMN-RBC system (Table 2) could

be reproduced by substituting catalase for RBCs in the

system (Table 3) and choosing an amount of enzyme (300

units in I mL) that results in a reduction of the HOC1

recovery from PMNs similar in magnitude to that observed

in the presence of 2 x 106 RBCs (Tables 2 and 3). The

requirement for 300 units catalase, ie, � 35 times the amount

of catalase in 2 x 106 RBCs, is conceivably due to the fact

that the enzyme is distributed throughout the assay volume

(I mL) whereas RBC catalase is confined to the cell pellet

(the HOC1 assays were carried out with cells as a pellet to

Table 3. Recovery of HOCI From OPZ-Triggered PMNs and/orMDCs in the Presence and in the Absence of Catalase

Assay Con�tions HOCI Recoveryt

PMNs

MDCs

PMNs + MDCs

PMNs + catalase

MDCs + catalase

PMNs + MDCs + catalase

15.6 ± 1.9

0

15.7 ± 1.3

8.8 ± 1.4

0

14.3 ± 1.0

PMNs, 5 x i0�; MDCs, 5 x iOn; catalase, (bovine liver, Sigma).

300 units.

tIn nmol/20 mm, � ± 1 SD of three experiments.

simulate the conditions used for measuring the lysis). Third,

the PMN-mediated RBC lysis was augmented by 5 mU/mL

GO, per se virtually ineffective (Fig 4). The lysis was

prevented by the MPO inhibitor azide as well as by the

HOC1-trapping agent taurine (Fig 4). In addition, 5 mU/mL

GO enhanced the recovery of HOC1 from the PMN-RBC

system by 46.9% + 5.2% (� ± 1 SD, n = 3), against an

expected value of 55% (value calculated on the basis of the

regression line from Fig 3 by using the extent of the RBC

lysis by PMNs plus GO as known variables).

DISCUSSION

In our model system as reported elsewhere6 and herein

confirmed by probing cytolysis with chemical agents, the

RBC lysis by PMNs requires HOC1 production via the

MPO-catalyzed oxidation of chloride ions by H202.

Although the cytolysin or cytolysins actually operative

remain to be identified,”�#{176}’2’ the extent of the lysis strictly

correlates with the amount of HOC1 generated by PMNs, at

least under our experimental conditions (the present paper

and Dallegri et al6). Consistent with this view, MPO-

negative MDCs incapable of generating HOC1 did not cause

lysis unless supplemented with MPO activity from a super-

40

30

Fig 3. Positive linear relationship (P < .01) between the RBClysis mediated by PMNs (#{149})and PMNs plus MDCs (�) (data fromFigs 1 and 2) and the HOCI recovery (nmol/20 mm) from PMNs (#{149})and PMNs plus MDCs (U) in presence of RBC targets (as for thelytic assays. the number of RBCs was kept constant. 2 x 10). Eachpoint represents � ± 1 SD of at least three experiments. Regres-sion line, y - 5.436 x -31.266.

Fig 4. Effect of GO on the RBC lysis by OPZ-triggered PMNs.The ordinate shows the percent lysis by (A) 0.25 x 10’ PMNs. (B) 5mU/mi GO. (C) 0.25 x i0� PMNs plus 5 mU/mL GO. (D) 0.25 x 10’PMNs plus 5 mU/mi GO plus 1 mmol/L azide. and (E) 0.25 x 10’PMNs plus 5 mU/mi GO plus 20 mmol/L taurine. The number ofRBCs is 10’/welI. Results are expressed as � ± I SD of threeexperiments.

For personal use only. by guest on July 10, 2011. bloodjournal.hematologylibrary.orgFrom

MDCS ENHANCE RBC LYSIS BY PMNs 1747

natant of the PMN granule fraction (unpublished observa-

tions). Nevertheless, they efficiently augmented both HOC1

recovery from the PMN-RBC lytic system and the HOC1-

dependent lysis.

A series of interconnected events underlies the expression

of the observed MDC “helper” activity. After the interaction

between the PMN surface and OPZ, a plasmalemma-

associated NADPH oxidase system is activated in the area of

PMN-OPZ contact,22 with consequent H202 production. The

generated H202 is released directly into the extracellular

fluid until the OPZ particle is completely engulfed. Part of

this H2O2 can probably undergo backdiffusion into PMNs

themselves.23 At the luminal surface of the phagosomal

membrane, which is derived from the plasmalemma, H202

production continues,24 and the generated H202 can diffuse

into PMN cytoplasm and outside the cell.25 The net amount

of H202 released extracellularly (extracellular H202 pool)

represents that fraction of H202 synthesized and not used by

PMN catabolic pathways in different subcellular compart-

ments, including MPO within phagosomes5’2’ and the cata-

lase and glutathione cycle within the cytosol.2�28 Although

discrepancies exist among different authors,’3’29 in our set-

ting the extracellular H2O2 pool is likely to be transformed

entirely into HOCI by the MPO released into the medium or

absorbed to the PMN surface. In fact, HOCI production by

OPZ-triggered PMNs could not be augmented by adding an

appropriate surplus of MPO activity, which suggests that the

H2O2 available for the chlorinating reaction is entirely used

by the released MPO. Moreover, HOC1 production could not

be augmented by biochemical (GO) or cellular (MDCs)

extra sources of H2O2, which suggests that the amount of

MPO released functions at a maximum rate without a

potential reserve of activity. Thus, it appears that OPZ-

triggered PMNs produce a definite amount of HOC1 by

releasing MPO and H2O2 via a process so balanced that

excesses of MPO and H202 are virtually absent or, if present,

trivial at least under our conditions (pH, C I concentration,

temperature). Although the H202 released and transformed

into HOCI represents a relatively small fraction of the total

amount of H202 produced (Nauseef et al,26 Test and Weiss,27

and unpublished observations) and is generally assumed to

be in dynamic equilibrium with the remainder (primarily

dissipated by the PMN catabolic pathways mentioned ear-

her), it can be expected that the addition of extra H202-

consuming pathways to the PMN microenvironment can

perturb the equilibrium of the system so much that subse-

quent HOC1 production is impaired. RBCs added to OPZ-

triggered PMNs to make up a complete cytolytic system

reduce the HOCI production by consuming H202 primarily

via catalase (Dallegri Ct al,6 Test and Weiss,27 and the

present paper). Such a consumption of H202, which results in

a relative impairment of HOC1-dependent PMN lytic effi-

ciency,6 is likely to take place within RBC targets before

lysis. In fact, the PMN-delivered lethal hit and the RBC

catalase-mediated impairment of the lysis occur during the

first 45 minutes of incubation when target cells are still

“viable” (changes in the final extent of the lysis could not be

detected when exogenous catalase was added to block the

lytic pathway 45 minutes after the start of the incubation

period, lysis at this time being virtually absent; unpublished

observations). This is consistent with results obtained by

other authors in a different system�#{176}and with the fact that

H202, having a relatively low reactivity and no charge, can

diffuse away from its sites of formation and across cell

membranes easily.3’ Although attempts to directly measure

the actual size of the extracellular H202 pool in our cytolytic

system with cells as a pellet (without adding extra H202-

consuming pathways, for instance, HRP) are presently ham-

pered by the nonexistence of a reliable H202 assay, the

results herein reported provide inferential evidence that the

reduction of the PMN extracellular H202 pool by RBC

catalase and the reconstitution of this pool by MDCs are

crucial to MDC-mediated augmentation of PMN HOC1

production and, in turn, lysis. This is suggested by different

findings. First, neither HOC1 recovery nor the extent of the

lysis could be augmented by MDCs after inhibition of the

RBC H202-consuming capacity, ie, using RBCAT as targets.

Second, enhancement of HOC1 production by MDCs in the

PMN-RBC system could be reproduced by substituting an

appropriate amount of reagent catalase for RBCs in the

system. Third, MDC-dependent enhancement of both HOCI

recovery from the PMN-RBC system and PMN-mediated

lysis could be reproduced by replacing MDCs with an

appropriate H202-generating enzymatic system (GO, Fig 4)

or H202-generating, granule-depleted PMN cytoplasts

obtained as described by Roos et al32 and unpublished

observations). Fourth, the “final” events occurring in our

system in the presence and in the absence of MDCs (amount

of HOC1 produced and extent of the lysis) strictly correlate

(Fig 3). Thus, on the basis of these observations coupled with

the evidence that MDCs are per se cytolytically ineffective

and with the results obtained by probing the lytic systems

with chemical agents, we conclude that (a) MDCs amplify

the lysis by improving HOC1 production, presumably via an

effective restoration of the extracellular H202 pool generated

by PMNs and depleted by RBCs; and (b) if present, the

intervention of nonoxidative cytolytic factors, released by

MDCs and capable of contributing to the lysis,33 would be

trivial at least under the present conditions. In addition, the

involvement of PMN-directed cytokines released by MDCs

and capable of increasing the HOC1 production can be ruled

out because MDCs did not affect the HOCI recovery from

PMNs in absence of RBCs. Similarly, the possibility that

MDCs may act by releasing NH4�, which favors NH2CI

formation (NH4� + HOC1 -b NH2CI + H20 + � can

be ruled out because exogenous NH4� inhibited the lysis in

the PMN-RBC system thus suggesting that lytic levels of

NH2C1 cannot be reached in our conditions. Finally, because

MDCs had no effect when added to the PMN�RBCAI

system, it is unlikely that they act by sensitizing RBC targets

to the PMN-delivered, HOC1-mediated attack.

In conclusion, it has been reported that macrophages

ingesting effete PMNs or granules from PMNs become

capable of mediating MPO-dependent iodination.35 Further,

macrophages can use eosinophil peroxidase (EPO) to

enhance their microbicidal or cytolytic activity?�38 These

results have suggested a way by which MPO-negative and

MPO-positive phagocytes can interact synergistically in

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1748 DALLEGRI ET AL

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of MDCs as a model raises the possibility that macrophages

at inflammatory sites may interact with neutrophils to

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