Presenility of Granulocytes in DownSyndrome Individuals

Kozo Yasui,* Koji Shinozaki, Takayuki Nakazawa, Kazunaga Agematsu, and Atsushi KomiyamaDepartment of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan

Neutrophil function defects occur in indi-viduals with Down syndrome (DS). We ex-amined apoptosis of granulocytes (neutro-phils and eosinophils) in DS individuals andcontrol healthy subjects. Granulocyte sur-vival was shortened in DS individuals, andthe percentage of apoptotic granulocytesfrom DS during incubation was signifi-cantly higher than that from healthy sub-jects. The difference was time-dependent,and that between DS and healthy subjectswas nearly 30% after longer periods of incu-bation. In control granulocytes, both granu-locyte-macrophage colony-stimulating fac-tor (10 ng/ml) and interleukin-5 (5 ng/ml)counteracted the programmed cell deathand delayed the apoptosis caused by anti-Fas antibodies, whereas those inflammatorycytokines were not able to completely pre-vent cellular apoptosis in DS patients. Ap-optosis and functional impairment of granu-locytes may contribute to the risk of infec-tions underlying pathological conditions ofDS, and accelerated apoptosis of granulo-cytes may be a factor to prevent chronic air-way inflammation and bronchial asthma inDS individuals. Am. J. Med. Genet. 84:406–412, 1999. © 1999 Wiley-Liss, Inc.

KEY WORDS: neutrophils; eosinophils; ap-optosis; inflammation; granu-locyte-macrophage colony-stimulating factor; interleu-kin-5

INTRODUCTION

Down syndrome (DS) individuals have multiple im-munological and hematological abnormalities [Spina etal., 1981; Burgio et al., 1983; Lockitch et al.,1989].These associated disorders have been attributed to im-munodeficiency and increased susceptibility to acuteinfections [Spina et al., 1981; Burgio et al., 1983]. Inaddition, decreased microbicidal activity [Novo et al.,1993] and deficient chemotaxis [Khan et al., 1975;Novo et al., 1993] in neutrophil functions are observedin DS patients. Increased morbidity of DS patients re-sults from respiratory and gastrointestinal infectionswith a high incidence of heart failure and malignancy[Miller, 1971; Masaki et al., 1981].

Apoptosis is one of two major mechanisms for celldeath. Apoptosis has been distinguished from necrosisboth morphologically (cell shrinkage, cellular conden-sation, and nuclear pyknosis, showing no membranefracture) and histochemically (by propidium iodide dy-ing and DNA fragmentation) [Ellis et al., 1991]. Neu-trophils have the shortest half-life of all circulating leu-kocytes and after leaving the bone marrow are pro-grammed to die within 24 hr. This is a normalmechanism of clearance of aged neutrophils from a site ofinfection preventing further tissue injury [Cohen, 1991].Circulating bacterial products or endogenous cytokinesmay decrease the rate of neutrophil apoptosis, resultingin further tissue damage [Colotta et al., 1992; Lee et al.,1993]. Neutrophil functions are impaired in DS patients,and this may account for the high susceptibility to bac-terial infections in the patients [Whyte et al., 1993]. Wesuggest that the abnormalities in neutrophil function inDS are the result of accelerated apoptosis. To date, therehave been no studies of neutrophil apoptosis in DS indi-viduals. Fas antigen (APO-1; CD95), a member of thetumor necrosis factor/nerve growth factor receptor fam-ily, is capable of inducing apoptosis in susceptible cellpopulations [Itoh et al., 1991; Oehm et al., 1992]. It hasbeen shown that activated T cells can secrete a function-ally active soluble form of Fas ligand that is capable ofinducing apoptosis in other cells [Tanaka et al., 1995].The findings that neutrophils and eosinophils express thefunctional Fas antigen suggest that this molecule is in-volved in the regulation of inflammatory diseasesthrough removal of unwanted phagocytic cells [Iwai et

Contract grant sponsor: the Ministry of Education, Science,Sports, and Culture; Contract grant sponsor: the Ministry ofHealth and Welfare of Japan; Contract grant number: Grant-in-Aid for Scientific Research 09670797.

*Correspondence to: Kozo Yasui, M.D., Department of Pediat-rics, Shinshu University School of Medicine Asahi 3-1-1, Matsu-moto 390-8621 Japan. E-mail: koyasui@gipac.shinshu-u.ac.jp

Received 26 June 1998; Accepted 26 January 1999

American Journal of Medical Genetics 84:406–412 (1999)

© 1999 Wiley-Liss, Inc.

al., 1994; Matsumoto et al., 1995; Liles and Klebanoff,1995]. This has led to a hypothesis that the Fas-Fasligand system may serve as a pathological mechanismin systemic inflammation [Savill, 1997].

Here we report on the survival and apoptosis ofgranulocytes and on the influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) andinterleukin (IL)-5 on Fas-induced apoptosis in DS pa-tients with a comparison in age-matched healthy con-trols.

MATERIALS AND METHODSReagents

Percoll and dextran T500 were purchased from Phar-macia Fine Chemicals (Uppsala, Sweden). Endotoxin-free Histopaque, DMSO, Hepes, propidium iodide (PI),and the protease inhibitor leupeptin were obtainedfrom Sigma (St. Louis, MO). Recombinant human GM-CSF (rhGM-CSF) was supplied by Kirin Brewery (To-kyo, Japan), and human IL-5 was purchased from Gen-zyme Corp. (Cambridge, MA). IgM anti-Fas antibody(CH11), fluorescein isothiocyanate (FITC)-labeled anti-Fas monoclonal antibody (mAb), and FITC-labeledmouse IgG1 were purchased from MBL (Nagoya, Ja-pan). FITC-labeled anti-Bcl-2 (clone 124) was obtainedfrom Dako Japan (Kyoto, Japan). Anti-CD16 mAb-coated immunological magnetic beads, together withthe magnetic cell sorter system were purchased fromMiltenyi Biotec GmbH (Bergisch Gladbach, Germany).

Cell Isolation

Heparinized venous blood samples were obtainedwith informed consent from 10 DS patients (medianage, 35 years; range 21–48 years; 6 males and 4 fe-males) and age-matched healthy volunteers. All of theDS patients were cytogenetically diagnosed as trisomy21. At the time of the study, they had no infections, nocirculatory or hematological disorders, and no habitualuse of drugs. Neutrophils were isolated by dextransedimentation and centrifugation on a Histopaque gra-dient as described previously [Yasui et al., 1994a]. Con-taminating red blood cells were removed by cold hypo-tonic water lysis. The cells were subsequently sub-jected to another density cut using centrifugation overa Percoll gradient in order to isolate neutrophils fromcontaminating eosinophils. Eosinophils were purifiedfrom peripheral blood using gradient centrifugationand negative selection with anti-CD16 mAb-coatedmagnetic beads in a magnetic cell sorter. The purity ofthe isolate was assessed using prepared cytocentri-fuged smears and by staining with May-Grunwald-Giemsa (Merck, Darmstadt, Germany). Purities of neu-trophil and eosinophil preparations were >95%, andcell viability was >99%, as determined by trypan bluedye exclusion (Sigma).

Assessment of Apoptosis

May-Grunwald-Giemsa-stained cytospun smearswere prepared from cells incubated in HBSS bufferedwith 10 mM HEPES with 2% fetal calf serum (FCS),pH 7.35. The cells were incubated in the presence or

absence of anti-Fas antibody (CH11). The percentage ofapoptotic cells was determined from 500-cell counts bytwo observers in a blind fashion. The apoptotic cellswere identified by their characteristic chromatin con-densation, cytoplasmic vacuolization, and by the for-mation of apoptotic bodies. In the studies, the assaywas compared with PI dye exclusion. Granulocyte ap-optosis was quantified as the percentage of cells withhypodiploid DNA by flow cytometry. Apoptotic cellshave an intact membrane that excludes PI. The PI fluo-rescence of individual nuclei with an FL3 acquisitionwas plotted, and the data were registered on a loga-rithmic scale. An excellent correlation was found be-tween the morphological and flow-cytometry methods.

DNA Fragmentation

First, 2 × 106 cells were lysed by incubation for 15min in 400 ml of a cold mixture of 10 mM Tris-HCl (pH7.4), 0.2 mM EDTA, and 0.2 wt % Triton X-100. Thelysate was centrifuged at 10,000 × g for 5 min. Thesupernatant was extracted with chloroform/isoamyl al-cohol/phenol, and the aqueous phase collected. DNAwas precipitated with 50% 2-propranol and 0.5 M so-dium acetate and left at −80°C overnight. After diges-tion with 50 mg/ml RNase A (Boehringer-Mannheim,Germany) at 37°C for 1 hr, samples were electropho-resed through a 1.2% agarose gel and stained with 0.5mg/ml ethidium bromide.

Neutrophil Functions

Neutrophil motility was measured by the agaroseplate method [Yasui et al., 1994b], using 5 ml of 1.2%agarose, dissolved in HBSS containing Ca and Mg ions,and supplemented with 10% FCS (heat-inactivated) ina petri dish (Becton Dickinson, Lincoln Park, NJ). A10-ml aliquot of cell suspension containing 5 × 104 cellswas placed in the center well of the plate, and equalvolumes of chemoattractant [2 × 10−7 M N-formyl-methionyl-lencyl-phenylalanine (FMLP)] and HBSSwere placed in outer and inner wells, respectively. Af-ter incubation at 37°C in a 5% CO2 atmosphere for 2 hr,the plates were fixed and the cells were subsequentlystained with Wright stain (Sigma). Migration was de-fined as the linear distance that the cells moved fromthe center well in the direction of the well containingthe chemoattractant (chemotaxis). Superoxide anionproduction was determined at 37°C as a change in ab-sorbance at 550 nm from the baseline using a Hitachispectrophotometer U2000 [Yasui et al., 1994b]. The re-action proceeded for 2 min with FMLP (1 mM) andstopped by adding 0.5 mM Nethylmalemide. Superox-ide generation by 107 neutrophils was calculated byabsorbance change in the presence of 1 mM superoxidedismutase (SOD) and absence of SOD and by subse-quent dividing this value by 21.1 × 103 M/cm for themolar extinction coefficient.

Immunofluorescence Flow Cytometry

To determine Fas expression, cells were incubatedwith FITC-labeled anti-Fas antibody at 4°C for 30 min,washed twice with phosphate-buffered saline (PBS),and analyzed using a FACScan (Becton Dickinson). Fordetection of intracellular Bcl-2, cells were fixed and

Granulocyte Apoptosis in Down Syndrome Patients 407

permeabilized with Ortho Permeafix (Raritan, NJ) atroom temperature for 40 min. The cells were washedwith PBS at 4°C followed by the immediate addition ofFITC-conjugated antibody and further incubation at4°C for 60 min. The cells were resuspended in ice-coldPBS at a final concentration of 1 × 107 cells/ml. Stain-ing with nonimmunoactive FITC-labeled mouse IgG1was used as negative control.

Statistical Analysis

All data are presented as mean ± SD. The signifi-cance of differences between groups was determined byStudent’s t-test. A P value of <0.05 was considered sig-nificant in all cases.

RESULTSAnti-Fas Antibody-Induced Apoptosis

Neutrophils were treated with 1 mg/ml of anti-Fasantibody (CH11) for 8 hr. At the light microscopic levelas shown in Figure 1, neutrophils demonstrated char-acteristic apoptosis morphology: darkly stained pyk-notic nuclei shrank but cytoplasmic organelles re-mained structurally intact, and also cytoplasm vacu-olation were typical. The percentage of apoptotic cellswas higher in DS subjects than in the normal controls.Figure 2 shows that the mean percentage of apoptoticneutrophils in the control was 16.6 ± 3.2% withoutCH11 and 43.4 ± 3.6% with CH11, whereas that in DSwas 28.0 ± 2.5% (P 4 0.0025 versus control) and 57.1 ±6.8% (P 4 0.0437 versus control), respectively. Simi-larly, the mean percentage of apoptotic eosinophils inthe control was 23.9 ± 2.4% without CH11 and 44.8 ±2.1% with CH11, whereas that in DS was 30.8 ± 2.6%(P 4 0.0249 versus control) and 55.9 ± 3.0% (P 4 0.003versus control) for a 72-hr incubation, respectively.Thus, the survival of neutrophils as well as eosinophilwas significantly reduced in DS. Cellular apoptosis in-duced by anti-Fas antibody was confirmed by agarosegel electrophoresis (Fig. 3), showing a characteristicladder-like apoptotic DNA degradation pattern. TheDNA ladder pattern in eosinophils was quite similar(data not shown). A large difference (nearly 30%) in

neutrophil apoptosis between DS and healthy subjectswas observed without anti-Fas antibodies for a longerperiod of incubation (data not shown). Although prote-ase inhibitor (leupeptin) generally blocked cell death,augmented cell death was observed in this condition inDS (Fig. 4).

Functional Assessment of Neutrophils

We examined the effects of apoptosis on neutrophilcell functions and found neutrophil cell function sup-pression depending on the incubation time (Fig. 5). Incontrast, neutrophils under these conditions containedsimilar amounts of lysozyme. This finding indicatesthat the granules were condensed and preserved inapoptotic cells (data not shown).

GM-CSF- or IL-5-InducedGranulocyte Apoptosis

When neutrophils and eosinophils were incubatedwith GM-CSF (10 ng/ml, 45 pM), the percentage ofapoptotic cells decreased. After incubation for 8 hr withCH11 (1 mg/ml), approximately 50% of neutrophilsfrom the normal controls exhibited apoptotic character-istics. GM-CSF markedly prevented anti-Fas antibody-induced neutrophil apoptosis (from 46 to 18%, P 40.0047) (Fig. 6). Meanwhile, the prevention by GM-CSF of cell death in DS was lower than expected, and39% of neutrophils showed apoptotic characteristics (P< 0.001 versus control). When eosinophils were incu-bated for 72 hr, GM-CSF or IL-5 (5 ng/ml) also pre-vented CH11-induced eosinophil apoptosis in the nor-mal controls (from 46 to 25 or 26%, P 4 0.0025 or P 40.0039), whereas 36% of eosinophils showed apoptoticcharacteristics in DS patients (P 4 0.0245 or P 40.0183) (Fig. 6).

Expression of Fas and Bcl-2

Flow cytometry was used to analyze the cell surfaceFas expression and bcl-2 protein expression in granu-locytes. The mean fluorescence intensity for Fas ex-pression was not significantly different in DS and con-trol granulocytes. No difference in mean fluorescenceintensity for bcl-2 protein was observed between DS

Fig. 1. Morphological features of apoptotic neutrophils in DS patients (A) and controls (B). Neutrophils were incubated for 8 hr with 1 mg/ml anti-Fasantibodies. May-Grunwald-Giemsa stained; magnification, ×1,000.

408 Yasui et al.

and control eosinophils, and no decline in the proteinwas observed with or without stimulation by anti-Fasantibodies (data not shown).

DISCUSSION

Neutrophils and eosinophils are known to play cru-cial roles at inflammatory sites. As these granulocytesmaintain a numerical equilibrium between cell prolif-eration and cell-death, rapid numerical homeostasismay also play a critical role in the generation of aneffective inflammatory reaction. Our study demon-strated that granulocyte survival is shorter in DS pa-tients than in age-matched healthy controls. A directrelationship between apoptosis and loss of functions,i.e., phagocytosis and respiratory burst, has beenshown by varying proportions of apoptosis in neutro-phils [Whyte et al., 1993]. In order to prepare neutro-phils properly from peripheral blood and to measurerespiratory burst and chemotaxis, at least 3 hr shouldbe taken. This time may cause a significant reductionin neutrophil viability and functions. Functional de-fects of neutrophils observed in DS patients may thusbe related to shorter life span of their cells. The exactnature of shortened life span of granulocytes in DS

patients remains unclear. There is no difference in thegranulocyte surface Fas expression between DS andcontrol individuals. Bcl-2 protein is known to be animportant molecule that acts against several means ofapoptotic stimulation [Hockenbery et al., 1990; La-gasse and Weissman, 1994], but the action is not ex-pressed in neutrophils but in eosinophils [Iwai et al.,1994]. We could not find a clear difference in intracel-lular contents of bcl-2 protein between DS and controlsubjects either. Murphy et al. [1992] reported that in-terferon (IFN)-g and tumor necrosis factor (TNF) in-hibit IL-4-driven gd T-cell proliferation, but thymo-cytes of DS patients are more sensitive to inhibition byboth cytokines. It is plausible that these findings alsoimplicate apoptosis in the pathogenesis of DS, but theunderlying molecular mechanism of such connection isunknown. Murphy et al. [1992] proposed that gene(s)that interferes the sensitivity to cytokines is located onchromosome 21. The copper-zinc superoxide dismutase(CuZnSOD) gene resides on the chromosome and isoverexpressed in DS patients. CuZnSOD is a key en-zyme in the metabolism of oxygen free radicals. Highersusceptibility to apoptosis of thymocytes and bone-marrow cells is probably caused by increased oxidativedamages by a higher degree of peroxidation [Peled-

Fig. 2. Percentage of apoptotic granulocytes in Down syndrome patients and normal subjects (n 4 5). Neutrophils (n 4 10) and eosinophils (n 4 5)of DS patients were incubated for 8 hr and 72 hr, respectively. Cells were also treated with anti-Fas antibodies (CH11; 1 mg/ml).

Granulocyte Apoptosis in Down Syndrome Patients 409

Kamar et al., 1995]. Additional studies are necessary toelucidate the precise mechanism involved in the prese-nility of granulocytes in DS patients.

Several inflammatory cytokines, including GM-CSF,IL-3, and IL-5, have been reported to support the sur-vival and bring the activation of granulocytes [Lopez etal., 1986; Brach et al., 1992; Hu and Yasui, 1997]. Ap-optosis is now emerging as a major mechanism for safe

Fig. 3. Agarose gel electrophoresis of DNA extracted from neutrophilsafter culture for 8 hr. M, molecular weight markers; C, control neutrophils;D, neutrophils from DS patients; anti-Fas, neutrophils with 1 mg/ml anti-Fas Ab. DNA was extracted from equivalent cell samples.

Fig. 4. Agarose gel electrophoresis of DNA extracted from neutrophilsafter culture for 8 hr. M, molecular weight markers; C, control neutrophils;D, neutrophils from DS patients. Neutrophils were with 1 mg/ml anti-FasAb and were with leupeptin (50 mM) from the initiation of culture (PI).

Fig. 5. Effects of apoptosis (incubation time) on neutrophil functions.Neutrophils were incubated for various times, and functions were assessedas described in Materials and Methods. Superoxide production (107 neu-trophils) was determined by cytochrome C reduction assay (A). The actualleading front distance under agarose (chemotaxis) is the number × 0.375mm (B). Data represent the mean ± SD of three experiments, each con-ducted in duplicate.

410 Yasui et al.

clearance of unwanted cells during resolution of in-flammation [Lee et al., 1993; Savill, 1997]. These find-ings suggest that a variety of proinflammatory cyto-kines (GM-CSF, IL-3, IL-5) augment granulocyte sur-vival and activation and that the Fas-Fas ligandsystem inhibits them by inducing granulocyte apopto-sis and thus sedates the inflammatory reactions. It hasbeen thought that an essential feature of bronchialasthma is a persistent air-way inflammation. Inflam-matory cell infiltration is present in the airway wallseven in mild asthma [Sullivan et al., 1994; Beasley etal., 1989]. Bronchoscopic studies have demonstratedthat with proper therapy a late asthmatic reaction canbe prevented and a significant reduction in the numberof granulocytes in the airway can be achieved [Sullivanet al., 1994; Woolley et al., 1996]. Unnecessary prolon-gation of the granulocyte life and activation of granu-locyte functions may produce substances toxic to epi-thelial cells in the airway. We previously showed thattheophylline at a therapeutical concentration inducesapoptosis of cultured granulocytes [Yasui et al., 1997]and suggested that inflammatory reactions in the air-way could be regulated in part by granulocyte apopto-sis. There have been a few reports on the prevalence ofasthma in DS patients [Kjelmann, 1988; Forni et al.,1990]. The present study showed that expected granu-locyte survival was shorter in DS subjects than in age-matched healthy controls. In DS, granulocyte apoptosisis accelerated in various conditions. Unlike the healthycontrols, inflammatory cytokines (GM-CSF and IL-5)were unable to completely prevent cellular apoptosis inDS patients. Thus, it seems that acceleration of granu-locyte apoptosis is associated with the inhibition ofchronic airway inflammation and a low incidence ofbronchial asthma in DS.

In conclusion, profound loss of cellular functions as-sociated with programmed granulocyte cell-death maybe an important event in the regulation of inflamma-tion, promoting resolution rather than persistence oftissue injury. Induction of granulocyte apoptosis willprove to be a new therapeutic approach to inflamma-tory diseases.

ACKNOWLEDGMENTS

This work is supported in part by a Grant-in-Aid forScientific Research (09670797) from the Ministry ofEducation, Science, Sports, and Culture and from theMinistry of Health and Welfare of Japan.

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