CoxIL-10 Regulation of Neutrophil Apoptosis in Sepsis

Regulation of Apoptosis by IL-10 in the Resolution ofIn_ammatory Responses to Infection

Gerard CoxFather Sean O’Sullivan Research Centre, St. Joseph’s Hospitaland McMaster University, Canada

Initiation of the response to infection

[1] The host response to infection begins as a coordi-nated well-intentioned cascade of events, regulated inlarge part by cytokines [2]. At ~rst these are producedby macrophages but within hours adjacent structuralcells as well as recruited in_ammatory cells may be-come important contributors of pro-in_ammatory cy-tokines [3]. There are few examples of apoptosis at thisstage of the response. Two examples are the inductionof apoptosis in monocyte/macrophages infected withShigella [4] and hepatocytes infected with Listeria [5].This is accompanied by increased release of pro-in_am-matory cytokines (IL-1 and chemoattractants respec-tively) which may exaggerate the response. However,early in typical responses to common infections, thereare few data indicating the presence or relevance ofleukocyte apoptosis. On the other hand the rapid induc-tion of apoptosis in infecting organisms would be ex-pected to be bene~cial to the host. Such bactericidalapoptosis would be effective against extracellularpathogens but remains to be described.

Ampli~cation of the resonse to infection

with resolution

In most situations, the host response to infection leadsto clearance of organisms and subsequent clearance ofrecruited in_ammatory cells and eventual return tonormal of host structure and function. Such orderlyevents are controlled by anti-in_ammatory cytokinessuch as IL-10, IL-13, IL-4, TGF-b [1]. Soluble antago-nists such as IL-1 receptor antagonist and soluble TNFreceptor assist in the damping of in_ammation andfacilitate termination of the response once the infectingorganisms have been cleared. Such clearance of leuko-cytes should provide an excellent model of apoptosisenabling resolution. While evidence from human situ-ations has documented that recuited leukocytes do un-dergo apoptosis [6], the strongest experimental datashowing that this is relevant to the success of resolu-tion, are from animal studies. An important advantageof such experiments is the level of control the investi-gator has over the timing of the events. Since apoptosisis a rapid event, and in vivo is associated with promptef~cent disposal by engulfment [7], it is frustratinglydif~cult to quantify even in situations where it isknown that many cells are being cleared in situ. Thuswe exploited the animal model of acute, self-limited,

neutrophilic pulmonary in_ammation induced by intra-tracheal administration of LPS to the rat [8]. The timecourse and cytokine pro~le of this response are wellcharacterised [3], with development of neutrophilia be-tween 2 and 6h, and resolution occurring between 24and 72 h. By examining cells obtained by bronchoalveo-lar lavage at intervals between 6 and 72 h after chal-lenge, we were able to document the orderly sequenceof neutrophil in_ux, apoptosis of neutrophils and en-gulfment of apoptotic neutrophils by macrophages—Figure 1.

This sequence of neutrophil recruitment followed bydevelopment of apoptosis and subsequent clearance ofcells has been documented in another model [9]. Ishi etal. showed these events to occur in the recovery fromozone inhalation injury in rats. In human disease, wehave found neutrophil apoptosis and macrophage en-gulfment when examining cells obtained from sputumof patients with infective exacerbations of their under-lying chronic obstructive lung disease (unpublished ob-servations). However, there is considerable variationbetween subjects and within the same subject whensamples from different days are examined. We havefound no clear relationship yet, between the frequencyof apoptosis, absolute cell counts and frequency ofmacrophage engulfment. Woolley et al successfullyused a model of mild exacerbation of asthma due toreduction in inhaled steroid therapy to document eosi-nophil apoptosis during recovery upon reinstitution ofappropriate therapy [10]. It remains to be documentedthat such events occur in orderly fashion during reso-lution of responses triggered by infection in humans.

Ampli~ed response to infection without

resolution

The low-dose LPS model is an example of mild, self-limited lung injury. In particular there is little evidence

Supported by: Medical Research Council of Canada and OntarioThoracic Society

Address correspondence to: Gerard Cox, Firestone RegionalChest and Allergy Unit, St. Joseph’s Hospital, 50 Charlton Ave.E., Hamilton, Ontario L8N 4A6, Canada. E-mail:coxp@fhs.csu.mcmaster.ca

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Sepsis 1998;2:73–78

© Kluwer Academic Publishers. Boston. Printed in U.S.A.

of tissue disruption. In more severe septic insults andespecially where there is failure of resolution, injury tostructural cells is evident. In such events the systemicin_ammatory response may continue either with orindependent of, ongoing infection and may be termedthe Systemic In_ammatory Response Syndrome(SIRS). The perpetuation of such ampli~ed responsescan be thought of as the result of failure of the damp-ing, anti-in_ammatory systems or persisting aggres-sive pro-in_ammatory in_uences [11].

In their review, Bone et al. highlight observationsassociating morbidity with overactive anti-in_amma-tory in_uences which render the host immunode~centor anergic [1,12]. This has been termed the Compensa-tory Anti-in_ammatory Response Syndrome (CARS).There is now experimental evidence from in vivo stud-ies of both humans and animals [13] for this concept,that de~ciency of pro-in_ammatory cytokines is associ-ated with poor outcome. In certain patients, in whomnative production of IFN-c is low—perhaps due to highlevels of IL-10—treatment with IFN-c was bene~cial[14]. This suggests that interference with pharma-cologic doses of anti-in_ammatory cytokines may bedetrimental in the management of SIRS.

Role of apoptosis—structural cells

At the stage of inducing tissue damage in the course ofa septic response, it is likely that injured structuralcells die by apoptosis. An obvious cell of interest is thevascular endothelial cell which may be extensively in-jured during local or systemic infection. Such injurycontributes in large part to organ dysfunction that de-termines much of the morbidity and mortality associ-ated with severe infections. Endotoxin injury of endo-thelial cells is a commonly used model which has shownthat such injury leads to cell death by apoptosis [15].IL-10 can prevent apoptosis induced by some of themechanisms involved in these in vitro systems [16].Furthermore, it has been found to be effective in an invivo model of LPS-induced liver injury [17]. In thiswork IL-10 prevented apoptosis that usually occurs inliver cells following sensitisation with P. acnes and sys-temic administration of LPS as a model of sepsis.

Role of apoptosis—neutrophils

The recruited in_ammatory cells are supposed to un-dergo apoptosis and be cleared [18,19]. Neutrophils aretypically short-lived cells that die by apoptosis [20].However, their survival can be markedly prolonged bytreatment with a variety of agents including cytokinesthat are released in the course of in_ammatory re-sponses [21–24]. Prolongation of their lifespan wouldcontribute to their accumulation and persistence in tis-sues even if in_ux was terminated. This is an obviousscenario to expect bene~t from treatment with agentsthat antagonise survival and induce apoptosis. Againwe have used the rat-LPS model, which has the advan-tage of de~ned onset of the response, to demonstrate

Fig. 1. The pro~le over time, of neutrophil accumulation, de-velopment of apoptosis, and engulfment of apoptotic cell bymacrophages, that occurs during onset and resolution of anacute in_ammatory response in vivo. Rats received intratra-cheal injection of LPS 6lg and BAL was performed at inter-vals of 0 - 72 h. Panel A shows total counts of neutrophils re-trieved, peaking at 18h. Panel B shows total numbers ofapoptotic neutrophils present, with peak at 24 h. Panel C showsthe number of macrophages in which apoptotic neutrophilswere identi~ed in their cytoplasm. All outcomes were assessedat light microscopy of cytospins of cells obtained by BAL,stained with Diff-Quik. Reproduced with permission - Cox G, etal. Am J Respir Cell Mol Biol 19952:232–237.

74 Cox

this principle [25]. Based on preliminary ~ndings thatLPS-induced survival in neutrophils depended on pro-duction of an autocrine factor that inhibited apoptosiswe examined the effect of IL-10 in our model. Admini-stration of IL-10 simulataneously with LPS did notaffect the early phase of the response. Thus, TNF lev-els at 2 h and neutrophil numbers at 6 h were similar intreatment and control groups. However, at 24 h therewere much fewer neutrophils found in the treatmentgroup indicating that clearance of these cells occurredmore rapidly—Figure 2. When cells were obtained byBAL at 6h after challenge and cultured, there wasclear evidence of apoptosis occurring sooner in the neu-trophils from animals treated in vivo with IL-10. Alsowe could not further prolong the survival of cells ob-tained from control, LPS only-treated animals suggest-ing these cells were already stimulated/committed tosurvive by exposure to the in_ammatory mileu in vivo.This supports the concept that enhanced neutrophilsurvival contributes to accumulation and persistence ofthese cells during in_ammatory responses—which re-cently published work has demonstrated more directlyusing similar methods [26].

When IL-10 was added at later time points of 6 hand beyond, it had no effect on neutrophil numbersobtained by BAL at subsequent timepoints or on neu-trophil survival in culture ex vivo. If added to cellsobtained at BAL from LPS-treated animals it againhad no effect on neutrophil survival in culture. In ex-tension of this observation, we examined the effect of

IL-10 on other stimuli of neutrophil survival in vitro.Thus IL-10 was an effective inhibitor only of LPS ef-fects, and then only when administered with or soonafter LPS (manuscript submitted).

Neutrophil survival is increased in response to LPS-treated neutrophil supernatant, indicating productionof a soluble survival factor [25]. This effect was notaffected by IL-10. We take this to indicate that IL-10inhibits production of a range of new proteins by neu-trophils, and among these is the autocrine survival fac-tor. Taken with the data that IL-10 does not changesurvival of control, untreated cells, we believe thatIL-10 has an indirect anti-survival effect rather than adirect action to induce apoptosis. The relevance ofthese observations is that IL-10 may not be an effec-tive antagonist of stimulated neutrophil survival invivo, particularly in situations where the response, andthe cytokine production, have already become estab-lished.

IL-10 and other leukocytes

IL-10 is a potent inhibitor of cytokine production bymonocyte/macrophages [27]. This action is believed tobe the mechanism of its powerful protective effectsagainst mortality in animal models of sepsis [28,29].There is some evidence that IL-10 modulates survivalof peripheral blood monocytes (PBM) in vitro [30]. Incontrast to what we have found with neutrophils, addi-tion of IL-10 to untreated cells caused more rapid celldeath. Similar to our ~ndings, IL-10 did not affectstimulated survival (with GM-CSF or IFN-c).

In examination of the consequences of intracellularinfection with Salmonella, IL-10 was shown to be es-sential in maintaining macrophage viability [31]. In thiswork IL-10 prevented apoptosis of infected macro-phages but was not relevant to their bactericidal ac-tion. Different experience has been reported with in-tracellular infection by Listeria, where neutralisationof endogenous surface-bound IL-10 enhances bacterialkilling [32]. This inhibitory effect of IL-10 on listerici-dal function has also been shown in the CSF of infectedmice [33]. One clear conclusion from this diverse expe-rience is that we cannot safely generalise from anyobservation and the determination of how IL-10 modu-lates monocyte/macrophage survival and activationduring human infections remains to made.

Again there is con_icting experience with IL-10modulation of lymphocyte survival. Depending on howB cells have been activated IL-10 may act a growthpromoter or induce apoptosis (SAC pretreatment) [34].In a model of B cell lymphoproliferative disease, B-1cells from NZB mice, IL-10 was found to be a growthpromoting agent [35]. In a human study IL-10 inhibitedgrowth of B cell chronic lymphocytic leukemia (B-CLL)cells without inducing apoptosis [36]. However, hetero-geneity of response of B-CLL cells from different pa-tients, to IL-10 has been shown, including induction ofapoptosis [37]. IL-10 may be produced by Th-2 cells and

Fig. 2. Cotreatment with IL-10 enhances clearance of neutro-phils recruited to the lung during an in_ammatory response invivo. IL-10 1 lg, was co-administered with LPS 6lg by intratra-cheal injection, to rats. BAL was performed at intervals anddifferential cell counts carried out at light microscopy, usingcytospins stained with Diff-Quik. There was a statisticallysigni~cant reduction in neutrophils present at 18 h, denoted ★,p , 0.05. Reproduced with permission - Cox G. Am J Physiol199671:L566–L571.

IL-10 Regulation of Neutrophil Apoptosis in Sepsis 75

inhibit cytokine (IL-2, IFN-c) production by Th-1 cells[38]. Oddly, IL-10 can prevent apoptosis developing inIL-2-starved T cells, which appears counter-productive[39]. Lastly, in this area of immune activation, IL-10 hasbeen shown to strongly enhance apoptosis in dendriticcells in culture [40]. This effect, if it were to occur in vivomight be to dampen the primary immune response acti-vated by dendritic cells. In summary, we can speculateon the potential of IL-10 to regulate the immune re-sponse to infection by modulating apoptosis of key celltypes, but we really have no ~rm observations on sucha role in human conditions.

Other anti-in_ammatory cytokines

There are few data on the potential of cytokines otherthan IL-10 to regulate apoptosis of neutrophils duringrecovery from septic injury. While IL-6 is inducedearly in many responses its overall effect may be eitherto promote in_ammation or to enhance resolutionthrough the production of acute phase reactants (re-viewed in [41]). IL-6 plays a critical role in generatingappropriate responses to chronic infection. However, inclinical situations of severe infection, IL-6 levels in theserum correlate with mortality, suggesting to somethat this factor is potentially deleterious to the host[42]. We have recently examined neutrophil kinetics ina model of IL-6 de~cient mice [43]. In IL-6 knockoutmice, exposed to LPS either locally via inhalation ofLPS aerosol or systemically by intraperitoneal injec-tion, we found enhancement of lung neutrophilia andcirculating pro-in_ammatory cytokines respectively.Despite published evidence that IL-6 might induceneutrophils apoptosis [44], we found no signi~cant dif-ferences in rates of apoptosis in neutrophils obtained atBAL at different timepoints after challenge. This expe-rience indicates that the regulatory effects of IL-6(which may have the potential to regulate apoptosis) toreduce the in_ammatory response to LPS are directedat the cytokine cascade but not at the lifespan of neu-trophils.

Cytokines or other factors that induce

apoptosis in neutrophils

Tumor necrosis factor (TNF-a) induces apoptosis inneutrophils. However, it is a central regulator of theusual response to infections and it is unlikely thattreatment with TNF-a to induce neutrophil apoptosiswould prove advantagous to the host [2]. As mentionedabove, IL-6 may induce monocyte and neutrophil apop-tosis in vitro, but our experiments suggest this may notbe an important effect of IL-6 during in vivo responses[44].

There is clear evidence that activation of neutro-phils in certain circumstances may be associated withinduction of cell death—in contrast to the usual obser-vation that stimuli causing activation also postponedevelopment of apoptosis [21–23]. One convincing ex-ample of these events is the observation made in vitro,

that engulfment of E. Coli can rapidly induce apoptosisin neutrophils [45]. This was associated with stimula-tion of the respiratory burst and could be inhibited byantioxidants. Thus effective removal of infecting or-ganisms might be accompanied by simultaneousef~cient clearance of the engul~ng in_ammatory cells.This observation is somewhat at odds with the effect ofLPS—which might be derived from E. Coli duringinfections in vivo—to prolong neutrophil survival[22,25]. It will require careful observation of responsesto infection, and not just to LPS, to establish the rele-vance of these events in vivo.

Treatment with proteolytic enzymes—includingelastase, which could be released by activated neutro-phils—induces apoptosis [46] and following stimulationwith the protein kinase C agonist phorbol 12-myristate13-acetate (PMA), neutrophils die quickly [47]. ThisPMA-induced process of cell death is not typical ofeither apoptosis or necrosis but like that induced byengulfment of E. Coli, may be inhibited by oxygenradical scavengers. Again however it is not obvioushow such mechanisms, that require neutrophil activa-tion with degranulation or increased respiratory burst,might be exploited in vivo for the bene~t of the host.

Occupation and activation of Fas (CD95) on the sur-face of neutrophils induces cell death by apoptosis [48].It is plausible that increasing expression of Fas wouldbe a “natural method” of inducing apoptosis in neutro-phils. However, it appears that Fas is continuouslyexpressed and it is not down-regulated in cells stimu-lated to survive in vitro or in vivo [49]. As yet there areno sophisticated methods to increase presence of Fasligand selectively, to promote apoptosis, without caus-ing cell activation and potentially enhancing the in_am-matory response. Also surviving cells exhibit reducedsensitivity to induction of apoptosis via Fas. This hasbeen found with cells ex vivo, obtained from thein_amed lung in response to LPS [26]. However, cellsremain partially vulnerable to Fas so this strategy re-mains attractive but unproven as a therapeutic effortfor limiting the in_ammatory response to infection.

IL-10—Too much of a good thing?

While administration of IL-10 can protect the hostfrom a variety of insults including sepsis [27,28] andallergen challenge [50], there are examples of how ex-cess IL-10 might also damage the host by reducing theability to combat infection and clear organisms. Thereare data to suggest that excessive repression by IL-10might reduce levels of pro-in_ammatory (Th-1) cytoki-nes below threshold levels needed to facilitate repair[1,14,51]. Animal work, examining clearance of pneu-monia due to Chlamydia trachomatis in mice, showsthat high levels of IL-10, in certain mouse strains, wereassociated with reduced rates of clearance of infectingorganisms and consequently delayed resolution [52].Such defects were reversed by treatment with neutral-ising antibody to IL-10. These events are explained by

76 Cox

the fact that bacterial clearance in this model is a Th-1-dependent, delayed type hypersensitivity process, thatis susceptible to inhibition by IL-10.

It is now apparent that many cells in the body, in-cluding various white blood cells and structural cells,may be a source of IL-10. These different cell typesdisplay heterogeneity in how their production of IL-10is regulated. For example, the liver is a major sourceof IL-10 during cardiopulmonary bypass, and this isstimulated by corticosteroids [53]. Monocytes increasetheir production of IL-10 in response to LPS but this isinhibited by corticosteroids [27] while we have foundthat similar treatment has little effect on LPS stimu-lated IL-10 production by human bronchial epithelialcells (unpublished observations). We know that corti-costeroid treatment early in the course of severe sepsiscausing acute lung injury is ineffective overall, so atpresent it is not clear how to intervene to augmentIL-10 production without creating an additional risk tothe host.

Conclusions

Our understanding of the potential roles of IL-10 in theregulation of in_ammation due to infection is expand-ing rapidly [54]. The picture is not necessarily anyclearer with the addition of new information and seem-ing contradictions are evident in the literature. Withrespect to regulating the survival of leukocytes, I be-lieve IL-10 has a largely indirect mode of action toprevent the release of potential survival factors. Thesemight be produced by the cell itself, ie autocrine, or bereleased by other cells that are involved in the devel-oping in_ammatory response. Unlike the effects of Fasligand or TNFa, which directly induce apoptosis, theeffects of IL-10 are less easily predicted as they will bethe result of a change in the balance of active factors.Since mechanisms to induce IL-10 are still non-speci~c,we do not yet have the ability to exploit host produc-tion to prevent or abbreviate manifestations of disease.

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