coiling phagocytosis discriminates between different spirochetes … · myristate acetate, which...

INFECTION AND IMMUNITY,0019-9567/98/$04.0010

Feb. 1998, p. 627–635 Vol. 66, No. 2

Copyright © 1998, American Society for Microbiology

Coiling Phagocytosis Discriminates between Different Spirochetes andIs Enhanced by Phorbol Myristate Acetate and Granulocyte-

Macrophage Colony-Stimulating FactorM. G. RITTIG,1* J. C. JAGODA,1 B. WILSKE,2 R. MURGIA,3 M. CINCO,3 R. REPP,4

G. R. BURMESTER,5 AND A. KRAUSE5

Department of Anatomy1 and Department of Internal Medicine III,4 University of Erlangen, Erlangen,Max-von-Pettenkofer-Institut, University of Munich, Munich,2 and Department of Internal Medicine III,

University Hospital Charite, Berlin,5 Germany, and Department of Microbiology,University of Trieste, Trieste, Italy3

Received 14 July 1997/Returned for modification 5 September 1997/Accepted 2 October 1997

The mechanisms involved in coiling phagocytosis are not yet known, and it is not even clear whether thisphenomenon is either an incidental event or a specific response. Therefore, the phagocytic uptake of Borreliaburgdorferi and other spirochetes by human monocytes in vitro was used to investigate the involvement of bothsides—microbes and phagocytes—in coiling phagocytosis. As seen with electron microscopy, morphologicallysimilar Borrelia, Leptospira and Treponema strains induced markedly different frequencies of coiling phagocy-tosis. The monocytes used coiling phagocytosis for both live (motile) and killed (nonmotile) B. burgdorferi, butpseudopod coils were observed neither with fragmented B. burgdorferi nor with cell-free supernatant from B.burgdorferi cultures. Investigation of the relationship of coiling phagocytosis with other pseudopod-basedcellular mechanisms revealed that the use of bioreagents that inhibit conventional phagocytosis also inhibitedcoiling phagocytis but did not affect membrane ruffling. Bioreagents that increase membrane ruffling did notaffect phagocytosis of B. burgdorferi, except for granulocyte-macrophage colony-stimulating factor and phorbolmyristate acetate, which increased coiling phagocytosis selectively. These results demonstrate that coilingphagocytosis is not induced by microbial motility, viability, or a certain morphology and that it is not a randomevent. Rather, it is a selective uptake mechanism actively driven by the phagocytes. However, whether coilingphagocytosis represents an independent alternative to conventional phagocytosis or, alternatively, a fault inconventional phagocytosis remains to be determined.

The characteristic of coiling phagocytosis (23) is the appear-ance of pseudopod whorls, which are based on single folds ofthe phagocyte plasma membrane wrapping around microbes inmultiple turns. Coiling phagocytosis shares some features withmacropinocytosis and conventional phagocytosis (Fig. 1), suchas the use of single membrane folds (which is typical of mac-ropinocytosis) or the uptake of particulate material (which isthe function of phagocytosis); however, there also are differ-ences. In macropinocytosis, the single pseudopods eventuallybend back toward the cell surface, thereby randomly trappinglarge droplets of pericellular fluid which may accidentally in-clude particles as well. In conventional phagocytosis, particlesare specifically engulfed by pseudopods which are strictly mi-crobe apposed, since for this process receptors on the surfaceof the pseudopod have to interact directly with ligands on thesurface of the particle. In coiling phagocytosis, the single pseu-dopods do not trap fluid droplets but enclose microbes; how-ever, the multiple pseudopod whorls have largely self-apposedinstead of microbe-apposed surfaces.

Coiled pseudopods were randomly observed with a variety ofmicrobes (for a review, see reference 37), but further studies ofcoiling phagocytosis have concentrated on Legionella pneumo-phila and Borrelia burgdorferi. Our group could demonstratethat coiling phagocytosis of B. burgdorferi comprises severalunique events. In sequence, these are the participation of an

unilateral pseudopod, the nonappearing membrane fusion fol-lowing the first completed rotation of this pseudopod, theongoing rotation leading to pseudopod whorls with largelyself-apposed plasma membranes, the fusion and subsequentdissipation of the apposed membranes, and finally the nonly-sosomal degradation of the engulfed Borrelia within the cytosol(34–36, 38). These unique features not only are related toseveral basic aspects of phagocyte biology such as microberecognition, membrane turnover, and particle uptake and pro-cessing but also may contribute to the pathogenesis of Lymedisease. Therefore, insights into the mechanisms of coilingphagocytosis may shed light on unsolved problems in boththese fields.

However, progress in the work on coiling phagocytosis washampered by two major difficulties. First, the sequence ofevents involved in this process has been described phenotypi-cally, but the factors which promote coiling phagocytosis arenot yet known. It is not even clear whether coiling phagocytosisreflects either a microbial strategy, a specific response of thephagocyte, or a random event. Second, phagocytes use coilingand conventional phagocytosis as well as macropinocytosis si-multaneously for the uptake of B. burgdorferi, which makes theinterpretation of experimental effects difficult and requireselectron microscopy to distinguish between the different mech-anisms. The present study addressed these problems by tryingto classify coiling phagocytosis.

In this study, the possible role of microbial shape, motility,and viability in the onset of coiling phagocytosis was deter-mined by comparing the frequencies of coiling phagocytosis fordifferent spirochetes with similar morphologies and for viable

* Corresponding author. Mailing address: Department of AnatomyI, University of Erlangen, Krankenhausstrasse 9, D-91054 Erlangen,Germany. Phone: (49)913185-3707. Fax: (49)913185-2863. E-mail:[email protected].

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(motile) versus killed (nonmotile) Borrelia. Bioreagents whicheither inhibit conventional phagocytosis or stimulate mem-brane ruffling were tested for their possible effects on thefrequencies of coiling versus conventional phagocytosis. Theresults obtained thereby indicate that coiling phagocytosis of B.burgdorferi is initiated by heat- and aldehyde-insensitive moi-eties on the surfaces of the microbes and further regulated bysignals on the side of the monocytes. With granulocyte-mac-rophage colony-stimulating factor (GM-CSF) and phorbol 12-myristate 13-acetate (PMA), we found two substances whichincrease the frequency of coiling phagocytosis selectively.Apart from these results, the formation of surplus pseudopodswas noticed. The surplus pseudopods represent a hitherto un-recognized feature of coiling phagocytosis possibly caused bythe lateral spreading of intracellular signals which controlpseudopod extension.

MATERIALS AND METHODS

Reagents. The separation media Nycoprep, Polymorphprep, and dextran 500were obtained from Life Technologies, Eggenstein, Germany; PMA, N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), genistein, staurosporine, bisindolylmale-imide I-HCl (BIM), okadaic acid, and wortmannin were from Calbiochem, BadSoden, Germany; 4-bromophenacyl bromide (4BPB) was from ICN Biomedicals,Eschwege, Germany; recombinant human GM-CSF and basic fibroblast growthfactor (bFGF) were from R&D Systems, Bad Nauheim, Germany; endothelialcell growth supplement (ECGS) prepared from bovine brain, lipopolysaccharide(LPS) prepared by phenol extraction from Escherichia coli serotype O111:B4,N-formyl-Met-Leu-Phe (fMLP), 5-N,N-dimethyl-amiloride (DMA), bafilomycinA1 and sodium azide (NaN3) were from Sigma, Deisenhofen, Germany. Theremaining chemicals were from Merck, Darmstadt, Germany, and all cell culturereagents were from BioConcept, Umkirch, Germany.

Phagocytes and platelets. Peripheral blood leukocytes and platelets were iso-lated from leukocyte-rich plasma (generously provided by the blood bank at theUniversity Hospital of Erlangen) of healthy human blood donors. All preparativesteps were performed in polyethylene tubes at room temperature; Ca/Mg-freeDulbecco’s phosphate-buffered saline supplemented with 0.13% (wt/vol) sodiumEDTA and 1% (vol/vol) heat-inactivated fetal calf serum (FCS) was used forwashings and dilutions. Peripheral blood mononuclear cells (PBMC) and poly-morphonuclear cells (PMNC) were isolated by buoyant density gradient centrif-ugation procedures with the separation media Nycoprep for the PBMC andPolymorphprep for the PMNC, as specified by the manufacturer’s manual (30).The monocytes were further enriched by removing the CD21 lymphocytes from

the PBMC fraction via a rosetting step on ice with neuraminidase-treated sheeperythrocytes. The eosinophils were negatively enriched by removing the neutro-phils from the PMNC fraction by means of magnetic bead-coupled anti-CD16monoclonal antibodies in a magnetic cell sorter. All phagocytes were checked forviability by means of trypan blue exclusion, counted, resuspended in RPMI 1640culture medium containing 10% (vol/vol) heat-inactivated FCS (RPMI-FCS),and allowed to recover for 1 h at room temperature prior to the phagocytosisassay. Platelets were enriched from the leukocyte-rich plasma by removing con-taminating erythrocytes via sedimentation with dextran 500 and contaminatingleukocytes via centrifugation at low speed and were allowed to recover in RPMI-FCS at 37°C until use.

Spirochetes. Strains from six different Leptospira interrogans serovars (copen-hageni, icterohaemorrhagiae Bucker, grippotyphosa Mallersdorf, pomona,hardjo, and sejroe) from our own strain collections were grown in Ellinghausen-McCullough-Johnson-Harris leptospire culture medium. Treponema phagedenis(7) was cultured in thioglycolate broth. The eight B. burgdorferi sensu lato strains(B. burgdorferi sensu stricto, B. afzelii, and B. garinii; for details see Table 1 andFig. 2) and two strains of relapsing fever Borrelia (B. turicatae and B. parkeri [50])were cultured in BSK-H culture medium supplemented with 6% (vol/vol) heat-inactivated rabbit serum and 2 mM L-glutamine. Only highly motile spirochetesgrowing in log phase were used for the experiments. Prior to incubation, thespirochetes were washed in Hanks’ balanced salt solution, counted, and ali-quoted in RPMI-FCS.

Pretreatment of the spirochetes. Aliquots of B. burgdorferi sensu stricto LW2were killed by various physical and chemical methods (for details, see Table 2),the effectiveness of which was checked by light microscopy. Portions of thealiquots were either directly heated, illuminated with UV light, or treated with asonifier, or others were centrifuged first, and the B. burgdorferi pellets wereresuspended in either ethanol, glutaraldehyde, or p-formaldehyde. The alde-hyde-treated spirochetes were subsequently incubated with sodium borohydrideto block reactive aldehyde groups (27). The chemically killed microbes werewashed carefully and resuspended in fresh RPMI-FCS before being added to themonocytes, whereas this was not necessary for the physically killed microbes.

Pretreatment of the phagocytes. Aliquots of monocytes were stimulated byadding either GM-CSF (41), bFGF (32), ECGS as a crude extract of acidicfibroblast growth factor (28), PMA (22), fMLP (8), LPS (8), or autologousplatelets to the incubation medium. Enzymes were inhibited as follows: proteintyrosine kinases by addition of genistein (19), protein serine/threonine kinases bystaurosporine (19), protein phosphatases 2A and 1 by okadaic acid (20), andprotein kinase C by BIM (48). Other approaches were the inhibition of phos-phatidylinositol 3-kinase by wortmannin (2), phospholipase A2 by 4BPB (26),geranylgeranyltransferase I by AGGC (9), the Na1/H1 antiport by DMA (22),and the V-type H1-ATPases by bafilomycin A1 (22). In addition, monocytealiquots were killed by poisoning the cells with NaN3. All of these bioreagentswere administered 10 min prior to the incubation with B. burgdorferi sensu strictoLW2. Except for the monocyte aliquots treated with NaN3, which were carefullywashed before incubation, the substances remained in the incubation medium,and the concentration of each (for details, see Fig. 6) was readjusted to the finalvolume when the spirochetes were added.

Phagocytosis assay. For each experiment, 2 3 106 monocytes and 2 3 107

spirochetes, each in 0.5 ml of RPMI-FCS, were mixed in polypropylene tubes,giving a total incubation volume of 1.0 ml. In one experiment, pelleted monocytealiquots were resuspended in 1.0 ml of cell-free culture supernatant from B.burgdorferi sensu stricto LW2. Incubation generally took place at 37°C under 7%CO2 for 45 min and was stopped by adding cold Ito’s fixative (24) to the cellsuspensions. Usually, each experiment was performed in triplicate (some inquintuplicate) with monocytes from different individuals. The frequency ofphagocytosis observed with untreated monocytes for untreated spirochetes, re-flecting the spontaneous frequency of phagocytosis, was used as a control.

Electron microscopy. Following fixation for 4 h at 4°C, the specimens wereprepared for electron microscopy according to standard protocols (18). Briefly,they were postfixed with reduced osmium, encapsulated in agar, stained en blocwith uranyl acetate and phosphotungstic acid, dehydrated in a series of gradedethanolic solutions ending with pure acetone, and then embedded in Epon 812.Ultrathin sections were cut and placed onto 200-mesh standard square coppergrids, contrasted with uranyl acetate and lead citrate, and examined with a Zeisstype 906 transmission electron microscope.

Evaluation of the results. First, all experiments performed with the aliquotedmonocytes from the same individual donor were analyzed. Therefore, two dif-ferent ultrathin sections from each experiment and the control were scored in ablind fashion by counting the incidences of coiling and conventional phagocytosisfor at least 100 randomly chosen monocytes. Proportionally expressed, this gavethe frequencies of coiling and conventional phagocytosis. Then, the frequenciesof the control (reflecting the spontaneous phagocytic activity of the monocytes)was set at 100%, and the frequencies of the experiments were calculated pro-portionally to that of the control. This normalization of the values from thedifferent individuals allowed, in a second step, the comparison between thetriplicate or quintuplicate performances. The differences were analyzed by the x2

test, and a P of ,0.05 was considered to be significant.

FIG. 1. Schematic drawing of the morphological characteristics of macropi-nocytosis and of conventional and coiling phagocytosis. Single membrane foldswhich bend back toward the cell surface constitutes macropinocytosis; largedroplets of pericellular fluid (which may contain some particles) are randomlytrapped during this process. In conventional phagocytosis, particles are specifi-cally engulfed by circumferential pseudopods which are guided by direct recep-tor-ligand interactions with the particle. Characteristic of coiling phagocytosisare unilateral pseudopods repetitively rotating around particles, giving rise tolargely self-apposed pseudopod whorls. Depending on the microbes studied,pseudopod coils will transform either to ribosome-studded replicative vacuoles(23) or to organelle exclusion zones (34). The microbe-apposed membranes ofthe engulfing pseudopods are drawn bold.

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RESULTS

Morphologically similar spirochetes induce different fre-quencies of coiling phagocytosis. If coiling phagocytosis is trig-gered by a certain microbial morphology, then microbes withsimilar shapes should induce similar frequencies of coilingphagocytosis. Therefore, different strains of B. burgdorferisensu lato as well as relapsing fever Borrelia, Treponema andLeptospira strains, which all have an elongated, helical form,were compared for the frequency of coiling phagocytosis.

For all different strains of B. burgdorferi sensu stricto, B. ga-rinii, and B. afzelii, the frequency of coiling phagocytosis was inthe range of 40 to 60% of total phagocytosis, regardless of theirgenospecies, geographic or biological origin, or how often theyhad been subcultured (Table 1). Therefore, the mean of thesefrequencies was used for B. burgdorferi sensu lato (Fig. 3). Incontrast, considerable differences were seen in the rate ofcoiling phagocytosis among the other spirochetes investigated(Fig. 3). With regard to their frequencies of coiling phagocy-tosis, a pattern which placed the spirochetes in different groupsemerged. T. phagedenis was the only other spirochete whichinduced coiling phagocytosis almost as frequently as B. burg-dorferi (30 to 40%). B. parkeri and L. interrogans SV pomonareached about half the frequency of B. burgdorferi (10 to 30%).The next group, consisting of the L. interrogans SVs grippoty-phosa Mallersdorf, sejroe, and hardjo, showed a low frequencyof coiling phagocytosis (5 to 20%), whereas this uptake mech-anism was very occasionally found in the final group consistingof B. turicatae and the L. interrogans SVs icterohaemorrhagiaeBucker and copenhageni (1% or less). Obviously, the frequen-cies of coiling phagocytosis observed with the different spiro-chetes were not correlated to their evolutionary relationship(Fig. 2).

Coiling phagocytosis of spirochetes involves surplus pseudo-pods. The pseudopod coils were more pronounced with the

B. burgdorferi sensu lato species than with the other spiro-chetes. However, no differences between the different spi-rochetes were observed regarding the further fate of thepseudopod coils (Fig. 4E). Briefly, the pseudopod-microbecomplexes were moved into the monocytes, where the granularcytoplasms of the pseudopods gave rise to large organelleexclusion zones. The apposing membranes of the coiled pseu-dopods fused to electron-dense lines which subsequently dis-integrated. Although host cell vesicles, especially lysosomes,were not present in the vicinity of the spirochetes within theorganelle exclusion zones, the microbes were degraded.

A hitherto unrecognized feature, which was frequently ob-served with coiling phagocytosis of all spirochetes, was theformation of surplus pseudopods (Fig. 4A, C, and D). In asmuch as 20% of the pseudopod whorls, the most peripheralrotation of the coiled pseudopod was covered by an additionalpseudopod pointing in the opposite direction. These contraro-tating pseudopods did not contact the enwrapped spirochetesbut were apposing the pseudopod whorls. Both the initial coil-ing pseudopods and the surplus pseudopods were clearly dis-tinct from the funnel-like surface extensions which engulfedthe spirochetes in conventional phagocytosis (Fig. 4F).

Both viable (motile) and killed (nonmotile) microbes butneither fragmented B. burgdorferi cells nor culture superna-tant induce coiling phagocytosis. If highly motile microbeseventually rolled themselves into phagocyte pseudopods, thenonly live (motile) but not killed (nonmotile) microbes shouldinduce coiling phagocytosis. Therefore, monocytes were incu-bated with B. burgdorferi sensu stricto LW2 cells killed bydifferent chemical and physical methods (Table 2).

It was found that live (motile) and killed (nonmotile) B.burgdorferi cells were engulfed with similar frequencies of coil-ing phagocytosis. Furthermore, as long as the overall morphol-ogy remained intact, there was no obvious difference betweenthe different methods used to kill the microbes. In contrast,neither conventional nor coiling phagocytosis was observedwhen the microbes were disintegrated to small fragments byuse of a sonifier. In this case, the monocytes contained numer-ous spacious vacuoles filled with small particles of variable size,but it was not possible to unequivocally identify this material asspirochetal fragments. Phagocytic structures were also absentwhen monocytes were incubated with cell-free supernatantfrom B. burgdorferi sensu stricto LW2 cultures instead of themicrobes (data not shown).

To reaffirm that coiling phagocytosis was actively driven bythe phagocytes, monocytes were killed by blocking their oxi-dative metabolism with NaN3 before being incubated with vi-able microbes. In this experiment, B. burgdorferi cells still at-tached to the cell surfaces of the monocytes, but no spirocheteswere detected inside the killed phagocytes (Fig. 5A).

FIG. 2. Dendrogram showing the phylogenetic relationship of the differentspirochetes used in this study.

TABLE 1. Characterization of B. burgdorferi sensu lato strains used in this study

Genospecies Strain Passage no. Biological origina Geographicalorigin

Reference orsource

B. burgdorferi sensu stricto LW2 .200 Human ligament Germany 21B31 .200 Tick United States ATCC 35210ZS7 8 Tick Germany 42

B. afzelii PLe 10 Human EM Germany 51B29 .100 Tick Germany 43

B. garinii BITS .100 Tick Italy 11PBi 8 Human CSF Germany 51387 .100 Human CSF Germany 51

a EM, erythema migrans; CSF, cerebrospinal fluid.

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Surface membrane folds are necessary but alone not suffi-cient for coiling phagocytosis. If pseudopod whorls wereformed spontaneously by surface folds, then the frequency ofcoiling phagocytosis should parallel the frequency of mem-brane ruffles, and coiling phagocytosis should not be restrictedto a certain object. Therefore, the sections were screened forthe spontaneous formation of pseudopod whorls, and mono-cytes were pretreated with bioreagents known to enhancemembrane ruffling before being incubated with B. burgdorferisensu stricto LW2.

When looking for spontaneous coiling, we observed a totalof three empty pseudopod whorls in the several hundreds ofsections investigated (Fig. 5F). The growth factors bFGF andECGS, the chemotaxins fMLP and LPS, and particularly plate-let-rich serum enhanced membrane ruffling without affectingthe uptake of B. burgdorferi (Fig. 5E and 6). No matter howpronounced membrane ruffling was, no increase in the fre-quency of coiling phagocytosis was noticed with these sub-stances. In contrast, both the growth factor GM-CSF and thephorbol ester PMA not only stimulated membrane ruffling butenhanced the frequency of coiling phagocytosis between 1.5-and 2-fold, whereas conventional phagocytosis was not in-creased (Fig. 5D and 6).

To see whether the effect of GM-CSF was restricted to themonocytic lineage, neutrophils and eosinophils were tested inaddition (Fig. 7). With these two polymorphonuclear phago-cytes, the increase in the frequency of coiling phagocytosis was2.5-fold for the neutrophils and, most pronounced, 5-fold forthe eosinophils. Since the spontaneous frequency of coilingphagocytosis in untreated neutrophils and eosinophils wasmuch lower than for untreated monocytes (32 and 17% versus43%), in the end the frequencies following stimulation by GM-CSF were about the same for the three phagocyte populations(80 to 86%).

Coiling phagocytosis is closely related to conventionalphagocytosis but not to macropinocytosis. Coiling and conven-tional phagocytosis of B. burgdorferi are always observed simul-taneously with almost the same frequencies and can be distin-guished only at the electron microscopic level. This not onlymakes the evaluation of experimental effects laborious but alsosuggests a close relationship between these two uptake mech-anisms. To see whether coiling and conventional phagocytosiscan be separated, monocytes were pretreated with variousbioreagents which are known to inhibit phagocytosis beforeincubation with B. burgdorferi sensu stricto LW2.

The results of these experiments are shown in Fig. 5B and Cand 6. It was found that all of the phagocytosis inhibitorsequally decreased the frequencies of both coiling and conven-tional phagocytosis. With respect to the differences in efficacy,the substances can be classified in three groups. Blockade ofATP-requiring processes with bafilomycin A1, acidification ofthe submembranous cytosol with DMA, or inhibition of phos-phatidylinositol 3-kinase by wortmannin virtually abolished

phagocytosis; inhibition of protein tyrosine kinases by genisteinand blockade of geranylgeranyltransferase I by AGGC andphospholipase A2 by 4BPB reduced the frequency of phago-cytosis by approximately 50%; whereas inhibition of proteinserine/threonine kinases by staurosporine, protein phospha-tases 2A and 1 by okadaic acid, and protein kinase by BIMresulted in only about 25% reduction of phagocytosis. In gen-eral, these inhibitors did not affect the occurrence of mem-brane folds, except for the most effective substances, of whichbafilomycin A1 and DMA almost completely and wortmanninalso considerably inhibited macropinocytosis.

DISCUSSION

The aim of this electron microscopic study was to classifycoiling phagocytosis of B. burgdorferi by human monocytes,first by determining the role of the microbes and the phago-cytes in this process and second by characterizing its relation-ship with conventional phagocytosis and macropinocytosis, thetwo major pseudopod-based uptake mechanisms of the mono-cytes. The results obtained demonstrate that coiling phagocy-tosis is an active and selective process of the phagocytes, ini-tially triggered by heat- and aldehyde-insensitive moieties ofthe microbial surface. As far as the phagocytes are concerned,these results suggest that coiling and conventional phagocyto-sis are very closely related, most likely starting from the samephagocytosis-promoting receptor(s) which remain to be iden-tified. Apart from the insights into its mechanisms, this studyrevealed an additional feature of coiling phagocytosis whichhas not been recognized so far, namely, the involvement ofcontrarotating surplus pseudopods.

It has for long been known that various particles, even whenopsonized equally, are phagocytosed with different frequen-

FIG. 3. Frequencies of coiling and conventional phagocytosis observed withdifferent spirochetes. Human monocytes (2 3 106) were incubated with differentspirochetes (2 3 107) for 45 min, and the frequencies of phagocytosis weredetermined by electron microscopy as described in Materials and Methods. Thenormalized results are expressed as means 6 standard errors of the means ofresults from three separate experiments using monocytes from different donors,but the value given for B. burgdorferi sensu lato summarizes the mean values forthe eight different species tested (Table 1). For the differences between B.burgdorferi sensu lato and the other spirochetes, a P of ,0.05 was considered tobe significant (*). Although they are quite similar in morphology, the differentspirochetes induced totally different frequencies of coiling phagocytosis whichshow no correlation with their phylogenetic relationship. L.i., L. interrogans.

TABLE 2. Methods used to kill the Borrelia and the effect onfrequency of coiling phagocytosis by human monocytes

Tool Application Time Subsequentwashing

Coilingphagocytosis

Ethanol 70% (vol/vol) 15 min Yes UnaffectedGlutaraldehyde 2.5% (vol/vol) 15 min Yesa Unaffectedp-Formaldehyde 4% (wt/vol) 15 min Yesa UnaffectedLaboratory oven 80°C 5 min No Unaffected32-W UV bulb 1-cm distance 30 min No Unaffected30-W sonifier 20 kHz 15 s (twice) No Abolished

a Additional treatment with 1% (wt/vol) sodium borohydride for 10 min.



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FIG. 4. Electron micrographs showing the uptake of various spirochetes by human monocytes. Human monocytes (2 3 106) were incubated with various spirochetes(2 3 107; arrows) for 45 min. Pseudopod coils are shown for B. afzelii (A; bar 5 0.1 mm), T. phagedenis (B; bar 5 0.4 mm), L. interrogans SV icterohaemorrhagiae Bucker(C; bar 5 0.5 mm), and B. parkeri (D; bar 5 0.5 mm); in panels A, C, and D, the pseudopod coils are partly covered by contrarotating surplus pseudopods (arrowheads).In panel E, a pseudopod coil enwrapping an L. interrogans SV pomona organism has already been transformed to an organelle exclusion zone (asterisk) within thecytoplasm of the monocyte. The remnants of the fused pseudopod membranes are clearly visible (bar 5 2.0 mm). The pseudopod whorls are easily distinguished fromthe funnel-like, symmetrical surface extensions of conventional phagocytosis, shown in panel F for a B. burgdorferi sensu stricto organism (bar 5 0.2 mm). The asteriskin panel D indicates a spacious phagosome.

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FIG. 5. Electron micrographs showing the effects of various bioreagents on the phagocytosis of B. burgdorferi by human monocytes. Human monocytes (2 3 106)were pretreated for 10 min and then incubated with B. burgdorferi cells (2 3 107) for another 45 min. (A) Borrelia (arrows) are not found inside monocytes which hadbeen poisoned with 0.1% NaN3 (bar 5 1.0 mm). (B) Upon treatment with 10 mM bafilomycin A1, monocytes still extend membrane ruffles (asterisks) but do notphagocytose Borrelia (bar 5 2.0 mm). (C) Although 100 nM wortmannin almost completely inhibits phagocytosis, Borrelia (arrows) are occasionally internalized inspacious vacuoles (asterisk) reminiscent of macropinosomes (bar 5 0.4 mm). (D) GM-CSF at 100 ng/ml induces a dramatic increase in the rate of coiling phagocytosis(bar 5 0.3 mm). (E) Even abundant membrane ruffles of monocytes, as seen in the presence of 10% serum enriched with activated platelets, do not spontaneously formwhorls (bar 5 0.6 mm). (F) Empty pseudopod coils possibly indicating spontaneously coiled pseudopods, such as this one (asterisk) displayed by an untreated monocyte,are an extremely rare occurrence (bar 5 0.6 mm).

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cies, which has been attributed to their different morphologies(5). Possibly, differences in microbial morphology may accountnot only for different frequencies of one phagocytic mechanismbut also for the use of different uptake mechanisms such asconventional and coiling phagocytosis. In the present study,however, totally different frequencies of coiling phagocytosiswere noticed for morphologically similar spirochetes understandardized experimental settings, which rules out unequivo-cally that coiling phagocytosis is triggered by a certain micro-bial shape. It appears that morphologically alike Legionellastrains too induce different frequencies of coiling phagocytosis(reviewed in reference 13), and the importance of a certainmicrobial shape had previously been questioned by the mor-phological variety of the microbes which were randomly foundto be enwrapped by pseudopod coils (for a review, see refer-ence 37). However, the disparity of methods applied to theseformer studies limits comparative conclusions and may giverise to some debate.

Although not dependent on a certain morphology, coilingphagocytosis nevertheless is restricted to a minimum particlesize and is not triggered by soluble factors, as neither super-natant from B. burgdorferi cultures nor spirochetal fragmentsinduced this mechanism. Comparable results were obtained inthe Legionella model where whole bacteria but not liposomescontaining the major outer membrane protein of L. pneumo-phila induced coiling phagocytosis (3).

For trypanosomatides, it has been suggested that coilingphagocytosis results from the movements of the flagellum,rolling these protozoa into the membrane veils to which theyare attached (10, 45). This hypothesis was supported by thefinding that coiling phagocytosis was no longer observed with

glutaraldehyde-killed Leishmania (10). In contrast, both in thepresent study and in the Legionella model (6, 23, 33), the use ofeither motile (live) or nonmotile (killed) microbes did not alterthe frequency of coiling phagocytosis. A causative role of mi-crobial motility is also questioned by the fact that pseudopodcoils were randomly observed with motile as well as nonmotilemicrobes. However, this random nature of the previous obser-vations here again limits their use for comparative conclusions.A likely explanation for the contradictionary results for theglutaraldehyde-killed Leishmania (10) is the subsequent han-dling; we found it mandatory to treat aldehyde-killed Borreliacells with sodium borohydride before using them in the phago-cytosis assays; otherwise, the monocytes would not engulfthem.

The lack of difference between viable and killed microbesindicates that coiling phagocytosis is actively driven by thephagocytes and not by the microbes. This distinguishes coilingphagocytosis from nonclassical uptake mechanisms such as theforced endocytosis typical of apicomplexan protozoa (4, 44),the directed phagocytosis of some enteroinvasive bacteria (40),or the triggered macropinocytosis of Salmonella (16), whichall represent microbial invasion strategies. On the side of thephagocytes, coiling phagocytosis obviously is a regulated mech-anism, because the monocytes used it selectively for certainspirochetes, which is inconsistent with an accidental trappingof pericellular microbes. Accordingly, stimulation of mem-brane ruffling by bFGF, ECGS, or platelets was not accompa-nied by an increase in the frequency of coiling phagocytosis,and also the consistent observation of very few empty pseudo-pod whorls does not support the view that coiling phagocytosisis a spontaneous event. The mechanisms by which PMA and

FIG. 6. Effects of various stimulatory and inhibitory bioreagents on the frequency of coiling and conventional phagocytosis by human monocytes. Human monocytes(2 3 106) were pretreated with substances which either inhibit conventional phagocytosis or stimulate membrane ruffling. After 10 min, B. burgdorferi cells (2 3 107)were added for another 45 min; only the incubation medium containing NaN3 was replaced by normal medium. The frequencies of phagocytosis were determined byelectron microscopy as described in Materials and Methods; untreated monocytes gave the spontaneous phagocytic activity, set at 100%. The normalized results areexpressed as means 6 standard errors of the means of results for usually three (five for control, PMA, and GM-CSF) separate experiments using monocytes fromdifferent donors. All inhibitors of phagocytosis reduced coiling and conventional phagocytosis equally, regardless how strong their inhibitory effect was. Theruffling-stimulating or activating substances did not affect the uptake of Borrelia except for PMA and particularly GM-CSF, which increased the frequency of coilingphagocytosis selectively (*, P 5 0.05 by the x2 test) compared with the spontaneous uptake.



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especially GM-CSF increase the frequency of coiling phagocy-tosis remain to be determined, but considering the ratherprompt onset of this effect, the upregulation of receptorsand/or the activation of signal transduction pathways may beinvolved.

Although eukaryotic cells definitely display different types ofmembrane folds, it seems unlikely that coiling phagocytosis isthe result of a committed coiling pseudopod. Video monitoringrevealed that the engulfing pseudopods are not preexistent, asone would expect in this case, but are formed in reaction tothe attachment of the Borrelia (39), which suggests a role ofattachment-induced intracellular signals. Also, recent work in-dicates that different pseudopod-based activities of the phago-cytes involve the differential regulation of one pool of pseudo-pods rather than the existence of different populations ofpseudopods (2), and in conventional phagocytosis itself, mor-phological differences between phagocytic cups depend on thedifferent receptors involved rather than on different pseudo-pods (1, 46). Therefore, it is more likely that either a certaincoiling-promoting receptor or distinct intracellular coiling-pro-moting signals mediate pseudopod coils.

The phagocytosis-promoting receptor(s) for B. burgdorferihas yet to be determined. In the Legionella model, antibody-mediated blockade of the complement-receptors (CR) of types1 and 3 (CR1 and CR3) almost completely inhibited the ad-herence and subsequent uptake of L. pneumophila Philadel-phia 1 (29, 31). Since this particular microbe is exclusivelyinternalized via coiling phagocytosis (23), it would be naturalto assume that CR1 and/or CR3 is the putative coiling-pro-moting receptor if CR-mediated phagocytosis were not a clas-sical model of zipper-type phagocytosis. Moreover, the com-plement-fixing protein of L. pneumophila, the major outermembrane protein, induces conventional but not coiling phago-cytosis when incorporated into liposomes (3), and other Legio-nella strains induce conventional phagocytosis as well (12, 15,33). Obviously, the appearance of pseudopod whorls in CR-mediated phagocytosis is not the rule but the exception, sug-gesting the superimposing effect of a coiling-promoting factor.

It has long been known that most spirochetes, including B.burgdorferi (35), adhere in a polar orientation with their free

end in motion, which has been attributed to a polar clusteringof spirochetal adhesins upon contact with the host cells (14).An asymmetrical capping of microbial adhesins could result inan asymmetrical ligand-receptor interaction with an unilat-eral pseudopod. Alternatively, an asymmetrical clustering ofphagocytosis-promoting host cell receptors could have thesame effect. The view of coiling phagocytosis as an irregularityin conventional phagocytosis easily explains why all biore-agents which inhibited conventional phagocytosis also inhib-ited coiling phagocytosis. For B. burgdorferi, only plasminogenand urokinase-type plasminogen activator (uPA) have beenshown to cluster at either pole of this organism (25). Althoughboth the plasminogen and uPA receptors on the host cell donot mediate phagocytosis, the uPAR forms multimeric com-plexes with other membrane receptors, including CR3 (49),which do so. In such receptor complexes, these phagocytosis-promoting receptors may not be capable of guiding the enclos-ing pseudopods symmetrically.

Whereas this asymmetry hypothesis implies that pseudopodcoils result from the incapability of the phagocyte to formconventional cups, there is at least one example where thecoiling of pseudopods is a physiological event. In the nonen-docytic process of myelination, single membrane folds are usedby glial cells to enwrap axons, and the resulting pseudopodwhorls have axon-apposed as well as self-apposed membranefaces. Thus, the formation of pseudopod whorls may resultfrom a basic coiling-promoting program of metazoan cells,regularly used by glial cells to myelinate axons but occasionallyby phagocytes to engulf microbes.

In this respect, the observation that part of the pseudopodwhorls in coiling phagocytosis are covered by an additionalmembrane fold is interesting. In triggered macropinocytosis,the local outburst of overshooting pseudopods has been ex-plained by a lateral spreading of intracellular signals uponattachment of the Salmonella (17). The surplus pseudopods incoiling phagocytosis as well are never directly apposed to theengulfed spirochetes and therefore result from a lateralspreading of signals involved in the generation of unilateralpseudopods, rather than from a classical receptor-ligand inter-action. Although this may be the first time that surplus pseu-dopods have been remarked upon, these structures can beretrospectively seen in several previous publications not onlywith B. burgdorferi (34, 47), but also with T. brucei (45) and L.pneumophila (23), suggesting that they are a general charac-teristic of coiling phagocytosis.

In conclusion, the results of this study demonstrate thatcoiling phagocytosis is neither a random feature of membranefolds in general nor concomitant to other pseudopod-basedactivities of the phagocytes but rather a selective uptake mech-anism obviously triggered by microbial surface moieties. Basedon these results, the identification of the phagocytosis-promot-ing receptors for B. burgdorferi will be most helpful to deter-mine whether coiling phagocytosis reflects an intrinsic coiling-promoting program which diverts the pathway of conventionalphagocytosis, or whether pseudopod coils display a fault in thereceptor-ligand interaction during the onset of phagocytosis. Inthis respect, the identification of GM-CSF and PMA as coiling-promoting substances may be a clue as to the regulatory mech-anisms involved in coiling phagocytosis.

ACKNOWLEDGMENTSThis work was supported by the Bundesministerium fur Bildung,

Wissenschaft, Forschung und Technologie (grants 01 KI 9104 and 9503)and by the Deutsche Forschungsgemeinschaft (grant Sp 395/2-1).

We are indebted to Andrea Hilpert for skillful technical assistanceand to Tony Simpson and especially Helen Robey, London, England,for critically reading the manuscript and for helpful discussions.

FIG. 7. Effects of GM-CSF on the frequency of coiling phagocytosis by dif-ferent human phagocytes. Human monocytes, neutrophils, and eosinophils (2 3106) isolated from the same individual (different from those represented in Fig.6), were incubated with B. burgdorferi cells (2 3 107) for 45 min in the presenceor absence of GM-CSF (100 ng/ml) added 10 min before. The bars represent therelative frequencies of coiling and conventional phagocytosis with GM-CSF andwithout, determined by electron microscopy as described in Materials and Meth-ods, given as means 6 standard errors of the means of triplicate determinations.Essentially the same results were observed in two additional identically per-formed experiments with phagocytes from different donors, and the resultsshown are therefore considered to be representative. PMA and especially GM-CSF increase the frequency of coiling phagocytosis selectively. The stimulatedfrequencies of the different phagocyte populations peak at about the same level,although their spontaneous frequencies are quite different.



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