Immunology 1999 97 429–437

Lipopolysaccharide enhances FccR-dependent functions in vivo throughCD11b/CD18 up-regulation

C. RUBEL, P. MILIANI DE MARVAL, M. VERMEULEN, M. A. ISTURIZ & M. S. PALERMO Division Inmunologıa,Academia Nacional de Medicina, Buenos Aires, Argentina

SUMMARY

Fc receptors for immunoglobulin G (IgG) (FccR) mediate several defence mechanisms in thecourse of inflammatory and infectious diseases. In Gram-negative infections, cellular walllipopolysaccharides (LPS) modulate different immune responses. We have recently demonstratedthat murine LPS in vivo treatment significantly increases FccR-dependent clearance of immunecomplexes (IC). In addition, we and others have reported the induction of adhesion moleculeson macrophages and neutrophils by LPS in vivo and by tumour necrosis factor-a (TNF-a) invitro. The aim of this paper was to investigate CD11b/CD18 participation in LPS enhancingeffects on Fcc-dependent functionality of tissue macrophages. Our results have demonstrated thatLPS can enhance antibody-dependent cellular cytotoxicity (ADCC) and IC-triggered cytotoxicity(IC-Ctx), two reactions which involve the Fcc-receptor but different lytic mechanisms. In vitroincubation of splenocytes from LPS-treated mice with anti-CD11b/CD18 abrogated ADCC andIC-Ctx enhancement, without affecting FccR expression. Similar results were obtained withphysiological concentrations of fibrinogen. In this way cytotoxic values of LPS-splenocytesdecreased to the basal levels of control mice. Time and temperature requirements for suchinhibition strongly suggested that anti-CD11b/CD18 could modulate intracellular signals leadingto downregulation of FccR functionality. Data presented herein support the hypothesis thatfunctional and/or physical associations between integrins and FccR could be critical for themodulation of effector functions during an inflammatory response.

INTRODUCTION immune complex clearance.9 In addition, several authors havereported the induction of integrins on macrophages and neu-

Fc receptors for immunoglobulin G (IgG) (FccR) are involvedtrophils by LPS in vivo and by tumour necrosis factor-a

in the clearance of immune complexes, phagocytosis of anti- (TNF-a) in vitro.9,10body-coated pathogens, enhancement of antigen presentation, Integrins are a familiy of transmembrane glycoproteinsgeneration of reactive oxygen intermediates (ROI) and anti- that mediate cell–cell and cell–substrate interactions. Thebody-dependent cellular cytotoxicity (ADCC).1,2 These recep- subfamily of b2 integrins comprises four homologous heterodi-tors are particularly important in the immunological defence mers: CD11a/CD18 ( leucocyte-function associate molecule-1,against pathogens during infectious diseases.3,4 Opsonization LFA-1), CD11b/CD18 (complement receptor type 3: CR3,of pathogens with IgG and/or complement factors allows cells Mac-1), CD11c/CD18 (CR4, p150/95) and CD11d/CD18to recognize a wide variety of organisms using a limited (ad2).11 CD11b/CD18 recognizes a variety of exogenous andnumber of receptors. It has been widely demonstrated that endogenous substances, including complement componentGram-negative bacterial infection is a strong stimulus of iC3b, intracellular adhesion molecule-1 (ICAM-1), fibrinogen,mononuclear phagocyte functions and several reports have factor X, zymosan, b glucans, Escherichia coli andanalysed the in vitro effects of lipopolysaccharides (LPS) on Leishmania.12 In addition to the recognition phenomena, b2FccR.5 However, the results obtained are partial and contra- integrins mediate transmembrane chemical signalling anddictory, depending on experimental conditions and the cell transmit mechanical stress to the cytoskeleton.13,14 There ispopulations studied.5–8 We have recently demonstrated that increasing evidence that ligation of integrins and Fc receptorsin vivo LPS inoculation markedly enhanced Fcc-dependent is able to initiate complex transmembrane signalling pathways

contributing to the activation of several effector functions ofReceived 26 October 1998; revised 5 February 1999; accepted leucocytes such as the oxidative burst15 and degranulation,16

5 February 1999. which are required for effective killing of the target. Moreover,it was suggested that although CD11b/CD18 does not induceCorrespondence: Dr M. S. Palermo, Division de Inmunologıa,target cell lysis by itself, it could mediate an enhanced phago-Instituto de Investigaciones Hematologicas, Academia Nacional de

Medicina, Pacheco de Melo 3081 (1425), Buenos Aires, Argentina. cytosis via FccR.17,18

© 1999 Blackwell Science Ltd 429

C. Rubel et al.430

The aim of this paper was to investigate CD11b/CD18 released was expressed as the percentage of total radioactivity.Spontaneous release was always less than 5% and was sub-participation in the enhancement of Fcc-dependent func-

tionality by LPS. We further discuss the relevance of functional stracted to obtain specific cytotoxicity.and/or physical associations between integrins and FccR

Antibody-dependent cellular cytotoxicity (ADCC)for modulating effector functions during an inflammatoryADCC was assayed by the chicken red blood cells (CRBC)response.anti-CRBC system as previously described.20 Unless otherwisestated, cytotoxic assays were performed by reacting 1×106

MATERIALS AND METHODS splenocytes with 2×105 51Cr-labelled CRBC sensitized withsubagglutinating amounts of specific rabbit IgG.Animals and treatment

LPS from Escherichia coli O111:B4 was dissolved in 0·15 Non-specific immune-complex triggered cytotoxicity (IC-Ctx)NaCl in order to obtain a final concentration of 50 mg/ml.Precipitating immune complexes (IC) were prepared withEight to 10-week-old BALB/c mice were i.v. injected withrabbit IgG antiovalbumin (OA), isolated by affinity chroma-0·1 ml of this solution and 20 hr later were sacrificed and theirtography and chromatographically purified OA (Cappel Lab.,spleens used for cytotoxic studies.Detroit, MI ). Immune complexes were formed at the equival-ent zone, previously determined by a precipitating curve.ReagentsAntigen and antibody in a molar ratio Ag5Ab=551 wereLPS from Escherichia coli O111:B4, phorbol myristate acetateincubated for 1 hr at 37° and 18 hr at 4°. Then, IC were(PMA), zymosan A from Saccharomyces cerevisiae yeast (Zy),washed and resuspended. Unless otherwise stated, 20 ml ofcatalase (bovine liver, type V, 3090 U/mg protein) and bovinethese IC were added to 1×106 splenocytes 5 min beforefibrinogen were purchased from Sigma Chemical Companythe addition of 2×105 non-sensitized labelled erythrocytes(St Louis, MO)(CRBC ).

AntibodiesPMA-induced cytotoxicity (PMA-Ctx)

Purified monoclonal antibodies to CD11b/CD18 (M1/70·15Splenocytes (1×106) were incubated with different concen-

clon, rat IgG2b anti CD11b) azida free, utilized in functionaltrations of PMA to induce the respiratory burst of splenocytes,

studies was a generous gift of Dr Francoise LePault fromand 2×105 51Cr-labelled non-sensitized erythrocytes (CRBC).

Unit 1461 CNRS URA. Hopital Necker, 161 Sevres 75743,France.19 The F(ab∞)2 fragment of monoclonal antibody to Opsonized zymosan-induced cytotoxicity (OpZy-Ctx)CD11b/CD18 was prepared by pepsin treatment and protein Zymosan A was washed in saline solution and suspended inA purification using a Immunopure kit (Pierce Chemical Co., human serum to a concentration of 10 mg/ml. After 30 minRockford, IL). Purity of F(ab∞)2 was confirmed by reducing of incubation at 37°, serum was removed by centrifugationand non-reducing sodium dodecyl sulphate–polyacrylamide and opsonized Zymosan (OpZy) was washed five times withgel electrophoresis (SDS–PAGE) with Coomassie staining. saline solution. Finally, OpZy was resuspended in salineIsotype control of rat IgG2b (Immunotech, France) and rat solution to a concentration of 10 mg/ml. Different concen-monoclonal antibody against murine CD18 (Immunotech, trations of OpZy were added to 1×106 splenocytes 5 minFrance) used in functional studies were exaustively dialysed before the addition of 2×105 51Cr-labelled non-sensitizedto remove azide. Cytometric studies were developed using erythrocytes (CRBC ).rat monoclonal antibodies against murine FccR (CD32/CD16) (Pharmingen, San Diego, CA) and murine CD11b/ Immunofluorescence flow cytometryCD18 (Boehringer Mannheim, Buenos Aires, Argentina). Measurement of the expression of FccR and CD11b/CD18Fluorescein isothiocyanate-conjugated goat antirat inmuno- on splenocytes was carried out by indirect immunofluorescenceglobulins (FITC-goat antirat IgG) were purchased from flow cytometry. The splenocyte suspension (20 ml; 25×106Pharmingen. Fluorescein isothiocyanate-conjugated mouse cells/ml ) obtained from saline and LPS-treated mice wasmonoclonal antibody to mouse RFccII/III (CD32/CD16) washed with cool phosphate-buffered saline (PBS) and incu-and R-phycoerythrin-conjugated rat monoclonal antibody to bated with monoclonal antibodies (mAb) (20 mg/ml to 1×106mouse CD11b were purchased from Caltag Laboratories cells) for 30 min at 4°. Then, the cells were washed and stained(Burlingame, CA), and utilized in double staining assays with fluoresceinated antirat IgG for 30 min at 4°. After

two washes with PBS, the cells were resuspended in 0·4 mlCytotoxic assays of ISOFLOW (International Link, S.A., Buenos Aires,Cytotoxic assays were performed by reacting splenocytes sus- Argentina) and the fluorescence was analysed by FACScanpended in complete medium (RPMI-1640, 3% fetal calf serum, (Becton Dickinson, Mountain View, CA). Double stainingGibco BRL, Gaithersburg, MD), with the appropiate stimulus. assays (CD11b+/FccR+) were carried out utilizing fluoresceinNeutrophil contamination of splenocyte suspension was always and R-phycoerythrin-conjugated antibodies. The analysis waslower than 1%. Macrophage in vivo depletion with liposomes made on 20 000 events on each sample by using the Cell Questcontaining dichloromethylene biphosphonate (Clodronate) Program (Becton Dickinson).completely abrogated cytotoxic functions, ensuring that thesereactions were carried out by the macrophage population Statistical analysis

All data are presented as the mean±SEM. Comparisons(unpublished observation). After incubation for 18 hr at 37°,the culture plate was centrifugated and the radioactivity of between multiple groups were performed with one-way analysis

of variance () followed by Bonferroni t-tests.supernatants and pellets was measured. The mean of 51Cr

© 1999 Blackwell Science Ltd, Immunology, 97, 429–437

LPS enhances FccR-dependent functions through CD11b/CD18 up-regulation 431

RESULTS CD11b/CD18 involvement in Fcc-mediated functions

Considering that integrins have been postulated as co-LPS in vivo treatment up-regulates CD11b/CD18signalling molecules of several receptors,12 we investigated

It has been established that the process of integrin CD11b/CD18 involvement in the enhancement of Fcc-up-regulation involves translocation of presynthesized intracy- mediated functions by LPS. Splenocytes from LPS or salinetoplasmic pools to the cell surface.21 However, depending on treated mice were incubated with anti-CD11b/CD18 mono-the stimulus and the cell target assayed this enhanced clonal antibody simultaneously with target cells in order toexpression could be transient or sustained.22 In order to assay ADCC, as described in Materials and Methods. Underanalyse integrin induction on effector cells by LPS in vivo these experimental conditions, anti-CD11b/CD18 antibody didinjection, CD11b/CD18 expression was evaluated on spleno- not exert any modulatory effect on cytotoxicity. However,cyte surface after LPS treatment. Doses as low as 5 mg/mouse when splenocytes were preincubated for 1 hr with the mono-(250 mg/kg, i.v.) of LPS induced a marked increase in spleno- clonal antibody the enhancement of ADCC induced by LPScyte CD11b/CD18 fluorescence intensity 3 hr post LPS inocu- was abrogated by anti-CD11b/CD18 at every effector5targetlation and, this effect persisted for at least 24 hr (Fig. 1). ratio assayed (Fig. 3a). Serial dilutions of the antibody showed

We had previously demonstrated that IC-clearance and that total inhibition occurred at a concentration of 0·7 mg/mlADCC were markedly enhanced by LPS treatment, without (5 n) (Fig. 3b). The inhibition of ADCC from LPS-treatedany alteration in FccR expression.9 Then, LPS effects on the mice was not due to non-specific blocking of FccR since thedouble possitive cells (CD11b+/FccR+) were evaluated using F(ab∞)2 preparation was also able to inhibit ADCC. Moreover,direct immunofluorescence. Results shown in Fig. 2 demon- equivalent concentrations of isotype matched mAb rat IgG2bstrated that the percentage of such cell population was (isotype control ) and anti-CD18 had no effect on cytotoxicityincreased after LPS treatment, due to an enhanced expression (Fig. 3b). The requirement of a preincubation time suggested

the involvement of a metabolic process in anti-CD11b/CD18of CD11b.

FL1-H

100

(a)

Cou

nts

101

102

103

104

20

15

10

5

0

Saline LPS20

15

10

5

0

100

101

102

103

104

(b)

Mea

n of

fluo

resc

ence

400

600

200

0

Saline

LPS

1 3 24

Time (hr)

Figure 1. Effect of LPS on CD11b/CD18 expression on splenocytes.At 24 hr post LPS injection, mice were killed and their spleenswere excised. The cells were stained with specific mAb anti-CD11b/CD18 as described in Materials and Methods. (a) Representativehistograms of CD11b/CD18 expression on splenocytes from saline and LPS-treated mice are shown. Controls of isotype weredrawn in dotted lines. The ordinate and the abscissa represent the cell number and the fluorescence intensity, respectively. In (b),saline and LPS-treated splenocytes were excised at different times after LPS injection and stained with specific mAb anti-CD11b/CD18 as described in Materials and Methods. Each bar represents the mean±SEM of arithmetic mean of fluorescence offour mice. *P<0·05 compared to saline treated group.

© 1999 Blackwell Science Ltd, Immunology, 97, 429–437

C. Rubel et al.432

FccR

100

(a)

MA

C-1

101

102

103

104

Saline LPS

100

101

102

103

104

(b)

Saline

LPS

100

101

102

103

104

100

101

102

103

104

CD11b+

%=29±2

X=1500±16

%=32±7

X=2310±115*

FccR+

%=10±1

X=126±17

%=4±1*

X=124±35

CD11b+/FccR

+

%=18±1

XCD11b=1858±105

XFccR=107±15

%=27±6*

XCD11b=3888±124*

XFccR=90±5

Figure 2. Effect of LPS on double positive CD11b+/FccR+ population. At 24 hr post-LPS injection, mice were killed and theirspleens were excised. The cells were stained with specific mAbs anti-CD11b and anti-FccR as described in Materials and Methods.(a) Representative dot plots of CD11b+/FccR+ expression on splenocytes from saline and LPS-treated mice are shown. Theordinate (Fl2) corresponds to R-phycoerythrin-conjugated anti-CD11b and the abscissa (Fl1) corresponds to fluorescein conjugatedanti-FccR antibodies. In (b), data represent the mean±SEM of percentage of positive population and arithmetic mean offluorescence of nine mice.*P<0·0001 compared to saline treated group.

inhibition of ADCC. In order to address this issue, splenocytes pation of different lytic mechanisms in both Fc-dependentcytotoxic assays, the reactions were carried out in the presencefrom LPS and saline treated mice were preincubated with anti-

CD11b/CD18 monoclonal antibody for 60 min at 37° or 4°. of 15 U/ml of catalase. This enzyme has been reported toimpair H2O2-mediated lysis generated upon burst respiratoryIn these experiments a final concentration of 4 mg/ml anti-

CD11b/CD18 was used after which target cells were added stimulation by IC, but it does not interfere with the ADCClytic mechanism.24 The results of a representative experimentand the reaction was carried out at 37°. When the temperature

of preincubation with monoclonal antibody was 4°, there was are shown in Table 2. We also confirmed previous evidencethat ADCC does not involve postphagocytic events, sinceno inhibition of ADCC from LPS-treated mice (Table 1).

These results demonstrated that anti-CD11b/CD18 inhibition cytochalasin B was not able to inhibit the level of cytotoxicity(data not shown).was time and temperature dependent, suggesting the involve-

ment of an active mechanism.We investigated the possibility that FccR splenocyte mem- CD11b/CD18 involvement in PMA-cytotoxicity

brane expression was modulated by this anti-CD11b/CD18Considering that PMA is able to directly stimulate protein

monoclonal. Our results were consistent with previous data,23kinase C (PKC) without any membrane receptor participation,

where the binding of monoclonal anti-FccR was not modifiedwe incubated splenocytes from LPS- and saline-treated mice

by preincubation with anti-CD11b/CD18 antibody (data notwith different doses of PMA. Splenocytes from LPS-treated

shown).animals showed an increased PMA-cytotoxicity, although pre-

We then examined the effect of anti-CD11b/CD18 on otherincubation with anti-CD11b/CD18 was not able to inhibit this

Fcc-dependent function, such as non-specific immune-complexreaction in any of the doses evaluated (Fig. 4b). This was also

triggered cytotoxicity (IC-Ctx). ADCC and IC-Ctx are bothobserved for control mice.

strictly dependent on FccR, although they involve differentactivation pathways.24 The results showed in Fig. 4(a) demon-

CD11b/CD18 involvement in zymosan cytotoxicitystrate that IC-Ctx is also enhanced upon LPS in vivo treatment.Moreover, anti-CD11b/CD18 preincubation significantly Zymosan opsonized with human serum (OpZy) represents

yeast particles coated with complement fragments, predomi-diminished this enhancement. In order to confirm the partici-

© 1999 Blackwell Science Ltd, Immunology, 97, 429–437

LPS enhances FccR-dependent functions through CD11b/CD18 up-regulation 433

significantly, suppressed this enhancement at the highest doseof OpZy assayed.

ADCC-modulation by fibrinogen, a CD11b/CD18 naturalligand

Considering that fibrinogen is the natural ligand ofCD11b/CD18 on monocytes and neutrophils,26,27 the modu-latory action of this ligand on ADCC was evaluated.Physiological concentrations of fibrinogen, added in solutionto culture medium, were able to induce inhibition of ADCCcarried out by splenocytes from LPS-treated mice (Fig. 5) atall effector:target ratio assayed. However, normal ADCC wasonly inhibited at the higher effector5target ratio.

DISCUSSION

We have presented a pannel of cellular cytotoxic reactionsmediated by splenocytes which are markedly enhanced afterin vivo LPS-treatment. These included FccR-dependent and-independent functions. The enhancement on FccR-mediatedreactions could not be associated with a higher expression ofFccR on cell membrane.9 On the other hand, a marked andpersistent increase in b2-integrins has been observed, con-firming previous information obtained in neutrophils.10,13Considering that recent reports have postulated that b2-integrins might function in a cooperative mannerwith Fc-receptors,28 we investigated the involvement ofCD11b/CD18 in LPS enhancing effects on FccR-dependentfunctions.

While CD11b/CD18 is normally found in small amountson the macrophage–monocyte cell surface, a larger quantityexists as a cytoplasmic pool, which becomes inserted intothe plama membrane in response to the treatment of the cellsAnti-CD11b/CD18 (µg/ml)

0

(a)

% A

DC

C

2 4 6 8

(b)

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

80

10

Saline

LPS

Effector:target ratio

0 2.5 5 7.5 10

Saline

LPS

Saline+anti-CD11b/CD18

LPS+anti-CD11b/CD18

with a number of cytokines and lipids.29,30 Enhanced surfaceFigure 3. (a) Effect of anti-CD11b/CD18 on ADCC from saline (%) expression is often accompanied by conformational changesand LPS (&) treated mice. Splenocytes were incubated for 60 min at in the integrin itself31 resulting in the expresion of activation37° with or without anti CD11b/CD18 at different effector5target epitope mAb2432 and the induction of a high avidity bindingratios, using 0·5, 1 and 2×106 splenocytes for 2·551, 551 and 1051

state of CD11b/CD18.29,31,32 Neutrophils and macrophagesratios, respectively. After 18 hr of incubation at 37°, the cytotoxicitytreated with LPS in vivo or TNF in vitro dramatically increasewas measured as described under Materials and Methods. Each pointthe level of cell-surface CD11b/CD18 expression.10 We consist-represents the mean±SEM of nine animals per group. (b) Splenocytesently found that LPS in vivo treatment increased the level of(1×106) were incubated for 60 min at 37° without or with differentthis molecule not only in peripheral neutrophils but alsoconcentrations of anti-CD11b/CD18. Additional controls of the

F(ab∞)2 fragment of anti-CD11b/CD18 (6), isotype matched antibody in splenocytes. Moreover, FccR+ cells which express a low(#) and anti-CD18 antibody ($) are also shown. density of CD11b molecules, significantly increase their*P<0·0001, compared with ADCC from LPS-treated mice without expression after LPS treatment, resulting an enhancement inantiCD11b/CD18. †P<0·0001, compared with ADCC from saline double high possitive cells (CD11b+/FccR+). However,treated mice without anti-CD11b/CD18. enhancement in CD11b/CD18 splenocyte expression was

minor and happened later in comparison with polymorphonu-clear cell (PMN ) induction. Peripheral PMN showed maximalnantly iC3b, a ligand for CD11b/CD18 (CR3). When phago-

cyte CD11b/CD18 binds to iC3b on yeast, phagocytosis and expression of CD11b/CD18 1 hr post-LPS treatment (250%compared to untreated cells), and splenocytes showed a 100%degranulation are triggered because of simultaneous recog-

nition of iC3b via a CD11b I-domain binding site and spec- enhancement 24 hr post LPS injection. TNF-a has been impli-cated as the endogenous cytokine which mediates several LPSific microbial polysaccharides via a lectin site located at

the COOH-terminal I-domain.25 Taking into account that actions,33 and TNF-a in vitro induces CD11b/CD18 expressionon PMN surface.10 However, after in vivo injection of murineCD11b/CD18 was up-regulated by LPS, the lytic activity was

assayed on unsensitized erythrocytes, triggered by OpZy. The TNF-a, we could not obtain any splenocyte modulation ofthis molecule nor Fcc-dependent functions (data not shown).possible inhibition by anti-CD11b/CD18 antibody was also

analysed. As shown in Fig. 4(c), this function was significantly The difference in kinetics of CD11b up-regulation betweenPMN and splenic macrophages, as well as the differentialincreased by in vivo treatment with LPS. However, anti-

CD11b/CD18 antibody preincubation only slightly, but not effect of TNF-a on both cell types, could reflect differences in

© 1999 Blackwell Science Ltd, Immunology, 97, 429–437

C. Rubel et al.434

Table 1. Time and temperature-dependence in anti CD11b/CD18 inhibitory effect on ADCC

Treatment

Inoculated Anti-CD11b/CD18 Incubation time Incubation temperature % ADCC Inhibitionwith (4 mg/ml ) (min) (°C) n ( X±SEM) (%)

Saline − −60 37 7 17·7±1·4 –Saline + −60 37 9 12·9±1·3 27Saline + −60 4 5 18·4±1·9 –LPS − −60 37 9 50·6±3·6 –LPS + −60 37 9 17·7±4·4 65*LPS + −60 4 5 40·6±6·2 20LPS + 0 37 4 61·3±13·3 −20LPS + +60 37 4 59·5±10·1 −17

Splenocytes (1×106) from saline- or LPS-treated mice were incubated at 37° or 4° without mAb or with anti-CD11b/CD18 (4 mg/ml ), added60 min before (−60), simultaneously (0) or 60 min after (+60), the addition of 51Cr-CRBC. After 18 hr of incubation at 37°, the ADCC wasmeasured as described under Materials and Methods. Each point represents the mean±SEM of n animals per group. *P<0·001 compared toLPS-treated splenocytes without mAb.

the underlying mechanism, i.e. translocation of an intracellular fail to amplify ingestion of either IgG- or C4b-opsonizedtargets in response to stimulation by phorbol esters, cytokinesstore (PMN) versus the de novo synthesis (splenocytes). This

hypothesis remains to be tested. or Arg-Gly-Asp (RGD)-containing adhesive proteins.37,38Both results, taken together, strongly suggest that duringCD11b/CD18 binding to its counter-receptors (ICAM-1)

has been found to play an important role in leucocyte adhesion inflammatory situations enhancement of Fcc receptor-dependent functions is associated with up-regulation ofto endothelial cells34 and to modulate H2O2 production.35

Moreover, b2-integrins are phagocytic receptors that bind a CD11b/CD18.On the contrary, PMA-induced cytotoxicity, which is alsovariety of bacterial products, including LPS. This binding

to integrins activates intracellular signalling pathways that increased by LPS treatment, was not affected by anti-CD11b/CD18 antibody. These results would indicate that theco-ordinate and regulate a variety of cellular responses.36

Additionaly, CD11b/CD18 forms transmembrane complexes interaction of monoclonal antibody with the integrin act onthe earliest steps before PKC activation. Anti-CD11b/CD18with glycosylphosphatidyl inositol-linked membrane glyco-

proteins such as FccRIIIB (CD16), the uroquinase- also failed to inhibit OpZy-Ctx enhanced by in vivo LPS,indicating that anti-CD11b/CD18 monoclonal antibody didplasminogen activator receptor (CD87), or the LPS receptor

CD14,11,37 providing these surface-bound molecules with not disrupt the signalling pathway triggered by the simul-taneous recognition of iC3b via a CD11b I-domain and thetransmembrane signalling, and modulating their activities. In

this regard, inhibition of neutrophil phagocytosis by anti- polysaccharides via a lectin site.39The observed inhibition of LPS-enhanced Fcc-dependentCD11b/CD18 has been extensively reported.17,18,23 On the

other hand, little is known concerning anti-CD11b/CD18 functions by anti-CD11b/CD18 antibody could potentially beexplained by two different mechanisms. Firstly, antibody toeffects on tissue macrophages carrying out other effector

functions, specially under inflammatory conditions. Although CD11b/CD18, either whole IgG or F(ab∞)2 fragments, couldsterically interfere with FccR on splenocytes or down regulatephagocytosis, ADCC and IC-Ctx are all FccR-dependent

functions, the intracellular pathways and lytic mechanisms its expression upon co-capping. However, we could not obtaindownregulation of FccR after anti-CD11b/CD18 incubationinvolved in each reaction are quite different.24 In addition,

monocytes and tissue macrophages employ different molecular either at 4° (steric interference) or at 37° (down-regulationupon co-capping), evaluated by cytometric assays. Thesemechanisms from those used by PMN for the same activity.24

In this context, we report new and additional CD11b/CD18 results are in concordance with Annenkov et al.,40 who didnot find any direct evidence of cross-blocking between CD11bmodulatory action on effector immune functions. The data

presented herein strongly suggest that LPS enhances FccR- and FccR in the K562 cells. Second, b2 integrins couldparticipate as active co-signalling molecules which would affectdependent functions: ADCC and IC-Ctx, through the

up-regulation of CD11b/CD18. We demonstrated that anti- the cellular response to signals transmitted via Fc receptors.The results presented herein favour the interpretation thatCD11b/CD18 antibody inhibits FccR-dependent functions in

a temperature and time dependent manner. Anti-CD11b/CD18 b2 integrins activated by inflammatory mediators couldco-operate in transmitting intracellular signals triggered byinhibits splenocyte activities specially when previously ampli-

fied by a systemic inflammatory process, such as LPS in vivo FccR. Moreover, the ligation of CD11b/CD18 receptor withits specific antibody, or with the physiological ligand fibrino-treatment, which leads to the up-regulation of CD11b/CD18

cell-surface expression. However, basal cytotoxicity in control gen, could interfere with a positive signal transduced byactivated b2 integrins or trigger a negative signal initiated aftermice was not significantly modified. In this regard, PMN from

patients with leucocyte adhesion deficiency (LAD), expresing binding. Recently, Fukushima et al.41 reported evidence thata similar mechanism is involved in the activation of Na+/H+a b2 integrin deficiency, exhibit normal Fcc-mediated phago-

cytic function in the absence of stimulation. However, they exchange upon FccR-binding in neutrophils. Considerable

© 1999 Blackwell Science Ltd, Immunology, 97, 429–437

LPS enhances FccR-dependent functions through CD11b/CD18 up-regulation 435

Table 2. Effect of catalase on ADCC and IC-CTX from LPS andprogress has been made in the last few years in definingsaline treated miceheterogeneity for IgG receptors through their molecular clon-

ing. These studies make it apparent that FccR share structur-Saline LPSally related ligand binding domains, but differ in their

ADCC 21·25±1·65 49·50±5·8ADCC + catalase 20·25±3·96 59·25±9·76IC-CTX 18·00±2·25 58·70±4·00IC-CTX + catalase 4·00±1·6* 5·50±0·64*

Splenocytes (1×106) were incubated in presence or absence of15 U/ml of catalase with 2×105 51Cr-CRBC sensitized, with subagglut-inant amounts of specific rabbit IgG for the ADCC assay, or with IC(20 mg/ml ) for the IC-Ctx. After 18 hr of incubation at 37°, thecytotoxicity was measured as described under Materials and Methods.Each point represents the mean±SEM of five animals per group.*P<0·0001 compared to IC-Ctx in the absence of catalase.

transmembrane and intracellular domains.42 These structuraldifferences account for different cellular signalling pathways.In this regard, our results lead us to speculate that overexpres-sion and/or activation of CD11b/CD18 enable specific FccRto transmit signals through integrin molecules. Overexpressionof integrins on cellular membrane probably increases theproximity between these molecules, allowing for close physicalassociations.

To understand how integrins regulate these events, it iscritical to identify and characterize the intracellular signallingpathways activated by integrin–ligand interactions. In thisregard it has been described that ligand occupancy of b2integrins lead to multiple intracellular events such as inter-action with cytoskeleton43 and tyrosine phosphorylation.21

In conclusion, new and compelling evidence would indicatethat multiple and common receptor-dependent intracellularpathways can synergize or antagonize in order to regulatecell function. A better understanding of these processes couldcontribute to future manipulation of immune functions.

Figure 4. (a) Effect of anti CD11b/CD18 on IC-triggered cytotoxicity(IC-Ctx) from saline (%) and LPS (&) treated mice. Splenocytes(1×106) were incubated for 60 min at 37° without mAb or withdifferent concentrations of anti CD11b/CD18. After 18 hr of incu-bation at 37°, the lysis was measured as described in Materials andMethods. Additional controls of the F(ab∞)2 fragment of anti-CD11b/CD18 (+) and isotype matched antibody (#) are also shown.Each point represents the mean±SEM of seven animals per group.†P<0·001 compared to IC-Ctx from saline treated mice.*P<0·001compared to IC-Ctx from LPS-treated mice without anti-CD11b/CD18. (b). Effect of the anti CD11b/CD18 on PMA-triggeredcytotoxicity from saline (%) and LPS (&) treated mice. Splenocytes(1×106) were incubated for 60 min at 37° without mAb (−) or withanti-CD11b/CD18 (+, 4 mg/ml ). Different concentrations of PMAwere added 5 min before the addition of non-sensitized 51Cr-CRBC.After 18 hr of incubation at 37°, the lysis was measured as describedunder Materials and Methods. Each point represents the mean±SEMof eight animals per group. †P<0·01 (LPS versus saline). (c) Effectof anti-CD11b/CD18 on zymosan-triggered cytotoxicity from salineand LPS treated mice. Splenocytes (1×106) were incubated for 60 minat 37° without mAb (% saline, & LPS) or with anti-CD11b/CD18(4 mg/ml, # saline, $ LPS). Different concentrations of OpZy wereadded 5 min before the addition of non-sensitized 51Cr-CRBC. After18 hr of incubation at 37°, the lysis was measured as described underMaterials and Methods. Each point represents the mean±SEM of 11animals per group. †P<0·001 (LPS versus saline).

Anti-CD11b/CD1810

–6M

% P

MA

-CT

X

(b)

0

10

20

30

40

50

60

80

SalineLPS

70

Anti-CD11b/CD18 (µg/ml)

0

% IC

-CT

X

2 4 6 8

(a)

0

10

20

30

40

50

60

80

10

SalineLPS70

10–6

M 10–7

M 10–7

MPMA+–+–

OpZy (µg/ml)

10

% O

pZy-

CT

X

20 30 40 50

(c)

0

10

20

30

40

50

60

0

Saline

LPS

70

60

Saline+anti-CD11b/CD18

LPS+anti-CD11b/CD18

© 1999 Blackwell Science Ltd, Immunology, 97, 429–437

C. Rubel et al.436

phagocytosis in bone marrow-derived mouse macrophages bybacterial lipopolysaccharide: correlation with biochemical andfunctional parameters. J Immunol 133, 913.

6. S H. & D’ R. (1994) Regulation of intracellular PMNleukocyte Fc receptors by LPS. Cell Immunol 157, 525.

7. F S., F H.G. & A D. (1991) Activation of macrophagesfor ADCC in vitro: effects of IL-4, TNF, IFN-a/b/c and GM-CSF.Cell Immunol 135, 78.

8. L G. & S S.R. (1994). Temporal down regulation ofFccRIII expression and FccR mediated phagocytosis in humanmonocyte-derived macrophages (MDM) induced by TNF-a andIL-1b. J Leukocyte Biol 55, 702.

9. P M.S., A R F., F A G. & I

M.A. (1997) Fcc receptor-dependent clearance is enhanced follow-ing LPS in vivo treatment. Immunology 92, 536.

10. W R., F A., S C.W. & J H. (1994)Complement and TNF contribute to Mac-1 (CD11b/CD18) upreg-ulation and systemic neutrophil activation during endotoxemia invivo. J Leukocyte Biol 55, 105.

11. V V M., L T H., W C.L. et al. (1995). AEffector:target ratio

2.5

% A

DC

C

5 7.5 100

10

20

30

40

50

70

0

Saline

Saline+FG

90 LPS

LPS+FG80

60

100

†

novel leukointegrin adb2, binds preferentially to ICAM-3.Figure 5. Effect of fibrinogen (FG) on ADCC from LPS and saline Immunity 3, 683.treated mice splenocytes. Splenocytes were incubated for 60 min at 12. P H.R. & T R.F. III (1996) Integrins as promiscuous37° with or without FG (6 m) at different effector5target ratios. After transduction devices. Immunol Today 17, 209.18 hr of incubation at 37°, the cytotoxicity was measured as described 13. F M., P F.M. & L R S.M. (1993) Activationunder Materials and Methods. Each point represents the mean±SEM of human neutrophils induces an interaction between the integrinof 12 animals per group. *P<0·01, compared with ADCC from LPS-

b2 subunit (CD18) and the actin binding protein a-actinina.treated mice without FG. †*P<0·05, compared with ADCC from J Immunol 151, 3795.saline-treated mice without FG. 14. C E.A. & B J.S. (1995) Integrins and signaling

transduction pathways: the road taken. Science 268, 233.15. Z M.J. & B E.J. (1994) CR3 (Mac-1,

aMb2,CD11b/CD18) and Fcc-RIII cooperate in generation ofAlthough further in vivo investigation is necessary, it is interes-neutrophil respiratory burst. Requirement for FccRII and tyrosineting to speculate that the anti-inflammatory in vivo effects ofphosphorylation. J Cell Biol 125, 1407.the anti-b chain antibodies seen in various pathological con-

16. H T.W., D K.M., L N.J. et al.ditions44,45 could be explained by the inhibition of cytotoxic(1990) Phosphatidylinositol-linked FcRIII mediates exocytosis ofreactions. Finally, the results obtained with fibrinogen haveneutrophil granule proteins, but does not mediates initiation ofphysiological implications, considering that it is an importantthe respiratory burst. J Immunol 144, 1432.

acute phase protein normally present in plasma. The role of17. G I.L., G H.D. & B E.J. (1989) An immobile

fibrinogen in regulating leucocyte functions during inflam- subset of plasma membrane CD11b/CD18 (Mac-1) is involved inmation, remains an unexplored field of investigation. phagocytosis of targets recognized by multiple receptors.

J Immunol 142, 2352.18. D P.A., Z D. & P D.E. (1994) Different signalingACKNOWLEDGMENTS

pathways for CD18-mediated adhesion and Fc-mediated phago-cytosis. Response of neutrophils to LPS. J Immunol 153, 2137.The authors thank Nora Galassi, Norma Riera, Juan Portaluppi and

Antonio Morales for their excellent technical assistance and Dr Jorge 19. F C., G M.C. & L F. (1994). ExpressionGeffner for reviewing the manuscript. We also thank Fundacion de la of homing and adhesion molecules in infiltrated islets ofHemofilia and Academia Nacional de Medicina for the use of the Langerhans and salivary gland of nonobese diabetic mice.FACScan flow cytometer. This work was supported by a grant from J Immunol 152, 5969.Consejo Nacional de Investigaciones Cientıficas y Tecnologicas 20. P P. & P H. (1970) Contactual lysis of antibody-(CONICET) and Fundacion Alberto J. Roemmers and Antorchas. coated chicken red erythrocytes by purified lymphocytes. Cell

Immunol 1, 300.21. B J.P., S S.G., R J. et al. (1990) Constitutive

REFERENCES and induced phosphorylation of the a- and b-chains of theCD11/CD18 leukocyte integrin family. Relationships to adhesion-1. R J.V. & K J.-P. (1991). Fc receptors. Annu Revdependent functions. J Immunol 144, 191.Immunol 9, 457.

22. D E.C.A., B G.M., P M.A., C L.A. &2. W P.K., H A.L. & F M.W. (1994). Role ofR G.J. (1996) Selective induction of CD11a,b,c/CD18 andFcc receptors in cancer and infectious disease. J Leukocyte BiolCD54 expression at the cell surface of human leukocytes by55, 816.muramyl peptides. Cell Immunol 169, 294.3. F M.W., S L., G R.F. & G P.M. (1989)

23. B E.J., B J.F. & G H.D. (1988) MechanismCytotoxicity mediated by human Fc receptors for IgG. Immunolof inhibition of immunoglobulin G-mediated phagocytosis byToday 10, 92.monoclonal antibodies that recognize the Mac-1 antigen. J Clin4. S D.P. (1992) Macrophages in bacterial infections. In: TheInvest 81, 365.Macrophage (Eds C. E. Lewis & J. O. McGee), p. 215. Oxford

24. G M., G J., S G., P M.S. &University Press, New York.5. C P.H., M P. & B M. (1984) Stimulation of I M. (1988) Different requirements for the induction of

© 1999 Blackwell Science Ltd, Immunology, 97, 429–437

LPS enhances FccR-dependent functions through CD11b/CD18 up-regulation 437

antibody-dependent and immune complexes triggered cytotoxicity lar matrix proteins and CD11/CD18 integrins. J Cell Biol 109,1341.mediated by monocytes. Immunol Lett 17, 109.

36. I R.R., A M.A. & G D.T. (1997)25. V V., T B.P. & R G.D. (1996) SolubleOutside-in signaling by lipopolysaccharide through a tailess inte-b-glucan polysaccharide binding to the lectin site of neutrophil orgrin. J Immunol 159, 433.natural killer cell complement receptor type 3 (CD11b/CD18)

37. G H.D., G I.L., A D.C. & Bgenerates a primed state of the receptor capable of mediatingE.J. (1991) Leukocyte adhesion-deficient neutrophils fail to ampl-cytotoxicity of iC3b-opsonized target cells. J Clin Invest 98, 50.ify phagocytic function in response to stimulation. Evidence for26. A D., B R., M P.M. & E T. (1988)CD11b/CD18-dependent and -independent mechanisms of phago-Oligospecificity of the cellular adhesion receptor MAC-1cytosis. J Clin Invest 88, 588.encompasses an inducible recognition specificity for fribinogen.

38. M T., O Y., N R., I A. & K T.J Cell Biol 107, 1893.(1993) Defective mononuclear cell antibody-dependent cellular27. B L., L J.D., K Z. et al. (1997) Mac-1cytotoxicity (ADCC) in patients with leukocyte adhesion(CD11b/CD18) is an oligodeoxynucleotide-binding protein.deficiency emphasizing on different CD11/CD18 requirement ofNature Med 3, 414.FccRI versus FccRII in ADCC. Cell Immunol 148, 385.28. W R.G., M-B L., W J.G.J.,

39. T B.P., V V., P M., G R.C. &T R.F III & P H.R. (1996) CR3 (aMb2; CD11b/CD18)R G.D. (1996) Analysis of the sugar specificity and molecularrestores IgG-dependent phagocytosis in transfectants expressing alocation of the b-glucan-binding lectin site of complement receptorphagocytosis-defective FccRIIA (CD32) tail-minus mutant.type 3 (CD11b/CD18). J Immunol 156, 1235.J Immunol 157, 5660.

40. A A., O S. & H N. (1996) The b2 integrin29. S J., M O., P W.F. et al. (1995) GranulocyteMac-1 but not p150,95 associates with FccRIIA. Eur J Immunolactivation via binding site near the C-terminal region of comple-26, 207.ment receptor type 3 a-chain (CD11b) potentially involved in

41. F T., W T.K., G S., G G.G.,intramembrane complex formation with glycosylphosphatidylinos-

O J. & D G.P. (1996) Na+/H+ exchange activityitol-anchored FccIIIB (CD16) molecules. J Immunol 154, 5452.

during phagocytosis in human netrophils: role of Fcc receptors30. D P.A., P D.E., W A., C-L I., and tyrosine kinases. J Cell Biol 132, 1037.

B M. & C Z.A. (1991) Differential effects of neutro- 42. D M. (1997) Fc receptor biology. Annu Rev Immunolphil-activating peptide1/IL-8 and its homologues on leukocyte 15, 203.adhesion and phagocytosis. J Immunol 147, 4211. 43. P F.M. & L R S.M. (1993) Activation of human

31. K P., P L. & J R. (1998). Integrins and GTPases neutrophils induces an interaction between the integrin b2-subunitin tumor cell growth, motility and invasion. Trends Cell Biol 8, 101. (CD18) and the actin binding protein a-actinin. J Immunol

32. S S.I., B A.R., T A.D. et al. (1995). -selectin 151, 3795.(CD62L) cross-linking signals neutrophil adhesive functions via 44. T E.I., S K., S S., C C. & Wthe Mac-1 (CD11b/CD18) b2-integrin. J Immunol 155, 1502. S.D. (1989) Reduction of inflammation, tissue damage, and

33. C A.S. & O S.M. (1995) Endotoxin’s role in Gram- mortality in bacteria meningitis in rabbits treated with monoclonalnegative bacterial infection. Curr Opin Infect Dis 8, 156. antibodies against adhesion-promoting receptors of leukocytes.

34. C-T E., S B., S C.W. & J Exp Med 170, 959.F J. (1995) Activation of human neutrophils through 45. B R.M., N-B L., B J.M., L V.C. &-selectin and Mac-1 molecules. J Immunol 154, 2291. S J.P. (1993) Mice treated with a leumedin or antibody

35. N C., S S., F C. et al. (1989) Cytokine-induced to Mac-1 to inibit leukocyte sequestration survive endotoxinchallenge. J Immunol 150, 3397.respiratory burst of human neutrophils: dependence on extracellu-

© 1999 Blackwell Science Ltd, Immunology, 97, 429–437