Haemophilus influenzae

Transcription

1 INFCTION AND IMMUNITY, June 1993, p /93/ $2./ Copyright X 1993, Amerian Soiety for Mirobiology Vol. 61, No. 6 Inhibition of Human Neutrophil Migration In Vitro by Low- Moleular-Mass Produts of Nontypeable Haemophilus influenzae D. R. CUNDLL,1* G. W. TAYLOR,2 K. KANTHAKUMAR,' M. WILKS,3 S. TABAQCHALI,3. DORY,4 J. L. DVALIA,S D.. ROBRTS,6 R. J. DAVIS,5 R. WILSON,' AND P. J. COL' Host Defene Unit, Department of Thorai Mediine, National Heart and Lung Institute, Manresa Road, London SW3 6LRA 1 Department of Clinial Pharmaology, Royal Postgraduate Medial Shool, London W12 CNN,2 Department of Medial Mirobiology,3 Department of Haematology, 4 and Aademi Unit of Respiratory Mediine,5 St. Bartholomew's Hospital, London C1A 7B, and Lilly Industries, Basingstoke, Hants, 6 United Kingdom Reeived 21 Deember 1992/Aepted 31 Marh 1993 Nontypeable Haemophilus influenzae ommonly auses infetions in the lower and upper respiratory trat, although the mehanisms of its olonization and persistene in the airways are unlear. Culture filtrates from six linial isolates of this baterium were assessed for their abilities to influene neutrophil funtion in vitro. ah ulture filtrate was assessed on six separate oasions with neutrophils obtained from six different donors. During the log and early stationary phases of growth ( to 18 h), ulture filtrates ontained primarily neutrophil hemokineti ativity but no ativity affeting neutrophil migration toward the hemotati fators N-formyl- L-methionyl-L-leuyl-L-phenylalanine and leukotriene B4. In ontrast, filtrates obtained after 24 h of ulture ontained fators whih inhibited neutrophil migration toward both of these hemotati fators. This hemotaxis-inhibitory ativity persisted between 24 and 72 h of baterial ulture, and it was not assoiated with the presene of either hemotati or hemokineti ativity as assessed by hekerboard analysis. Gel filtration of pooled 72-h filtrates yielded three major peaks of hemotaxis-inhibitory ativity. ndotoxin was present together with two other low-moleular-mass hydrophobi fators of approximately 8 and 2 kda. These low-moleular-mass fators are hloroform insoluble and heat stable, and they are inativated by protease, periodate, and diborane redution. Ativity was ompletely retained on a wheat germ agglutinin olumn, and it ould be eluted with N-aetyl-D-gluosamine. These data suggest that inhibitory ativity is assoiated with N-aetyl-D-gluosamine-ontaining glyopeptides, possibly derived from the baterial ell wall. The prodution of these ompounds may ontribute to the persistene of this baterium in vivo by inhibiting neutrophil hemotaxis in the miroenvironment of the respiratory muosa. Nontypeable Haemophilus influenzae (NTHi) is a normal nasopharyngeal ommensal, and arriage rates as high as 8% have been found in some ommunities (19). The baterium is also frequently assoiated with infetions of muosal surfaes, inluding middle-ear infetions, sinusitis, and infetive exaerbations of hroni bronhitis and bronhietasis (19). These infetions are assoiated with the rapid infiltration and subsequent ativation of polymorphonulear leukoytes (5). The most likely stimulus for this inflammatory response is the generation of endogenous and baterially derived hemotati fators (3, 13). In hroni diseases, these neutrophils, although ativated, fail to lear the organism, whih persists and spreads ontiguously in the airways. The mehanism by whih NTHi avoids elimination remains unknown, although several other gram-negative bateria, inluding Bordetella pertussis (1), Legionella midadei (7), and Pseudomonas aeruginosa (2, 1), all of whih ause infetions of the respiratory trat, have been previously shown to inhibit granuloyte funtions. The mehanisms are varied, but they are thought to involve enzymi inhibition of G proteins, aid phosphatase-indued blokage of membrane signal transdution, and ytotoxi effets (1, 2, 7, 1). In this study, we have investigated the effets of substanes produed by linial isolates of NTHi on the migra- * Corresponding author tion, surfae C3bi reeptor expression, and phagoyti apaity of human neutrophils, and we desribe novel lowmoleular-mass substanes apable of inhibiting the neutrophil response to the hemotaxins N-formyl-L-methionyl-L-leuyl-L-phenylalanine (FMLP) and leukotriene B4 (LTB4). MATRIALS AND MTHODS Preparation of baterial samples. Six strains of NTHi were isolated from the Sputasol-treated sputa of patients with hroni bronhitis, and they were identified by olonial morphology, Gram staining, and determining the requirement for fators V and X for growth aording to standard mirobiologial tehniques. ah of the six baterial isolates was ultured in Tryptiase soy broth (Unipath, Ltd., Basingstoke, United Kingdom) at 37 C (15) and sampled between and 72 h. Samples of the medium were entrifuged (5, x g, 15 min) and filtered through.2-,um-pore-size filters (Sartorius, Ltd., psom, United Kingdom) to produe baterium-free ulture filtrates for eah time point. Neutrophil isolation. Human polymorphonulear leukoytes were isolated from peripheral blood samples from human volunteers by dextran sedimentation and Fioll- Isopaque separation (Pharmaia, Ltd., Milton Keynes, United Kingdom) aording to the tehnique of Lee et al. (11). Neutrophils were washed (25 x g, 1 min) in Hanks

2 242 CUNDLL T AL. balaned salt solution (HBSS) (Sigma Chemial Company, Poole, United Kingdom), buffered to ph 7.4 with 3 mm N- 2-hydroxyethylpiperazine-N'-2-ethanesulfoni aid (HPS), and resuspended to a final onentration of 2 x 16 ells ml-1 in H3BSS ontaining.2% ovalbumin (Sigma). Neutrophils aounted for >95% of ells and were >98% viable as assessed by trypan blue exlusion and latate dehydrogenase estimation (Sigma). HBSS, HPS, and dextran were all prepared with endotoxin-free water (Sigma). Neutrophil migration. Baterial ulture filtrates were obtained from six different linial isolates of NTHi ultured for up to 72 h. For eah time point, the ulture filtrate from eah linial isolate was assessed separately. ah ulture filtrate was assessed on six separate oasions with neutrophils obtained from six different donors. Partially purified fators and the produts of hemial and enzymati ativation were similarly addressed. (i) Chemotaxis. Neutrophil migration was assessed over 9 min at 37 C in response to uninoulated ulture medium, HBSS alone, and dilutions of baterial ulture filtrates (undiluted, 1-') obtained from the six strains of NTHi between and 72 h by the leading front method in the modified Boyden tehnique (11). The positive ontrol was FMLP (1- M). The level of hemoattratant in a ulture filtrate was expressed as a perentage of the migratory response to FMLP. Chekerboard analysis was performed (2) with dilutions (1-' to 1-') of the 7- and 72-h samples of baterial ulture filtrates obtained from eah of the six linial isolates. (ii) Inhibition of migration. Neutrophils were pretreated either with dilutions (undiluted, 1-') of baterial ulture filtrates obtained at between and 72 h of ulture from the six strains of NTHi or with HBSS as the ontrol for up to 9 min at 37 C. They were then washed twie in HBSS by entrifugation (25 x g, 1 min) and resuspension, and their migratory responses to FMLP (1-9 M) were determined by the modified Boyden tehnique (11). In separate experiments, to investigate the reversibility of inhibition of migration, neutrophils were exposed to 72-h ulture filtrates (1-2 dilution in HBSS for 2 min, 37 C) obtained from eah of the six strains of NTHi, washed twie (25 x g, 1 min), and allowed to stand at room temperature in HBSS for either 3 or 6 min prior to assessment of their migratory responses to FMLP (1-9 M). Inhibition was expressed as the perent redution in migration toward the hemotaxin. Neutrophil phagoytosis and C3bi reeptor expression. Neutrophils were preinubated (2 min, 37 C) with uninoulated ulture medium, dilutions of 7- and 72-h baterial ulture filtrates (1-1 to 1-4) obtained from eah of the six strains of NTHi, FMLP (1-3 M; Sigma), or HBSS alone. They were washed twie in HBSS by entrifugation (25 x g, 1 min) and resuspension and were resuspended in HBSS-ovalbumin to a final onentration of 2 x 16 neutrophils per ml. Phagoytosis was assessed by light mirosopy under oil immersion with an opsonized latex bead model of ingestion (with gentian violet staining to test for the presene of beads) aording to a method previously desribed by Cline and Lehrer for marophages (4). Opsonized beads (1.3,um in diameter; Sigma) were prepared by inubation (overnight at 4 C) with fetal alf serum (ICN Flow, High Wyombe, United Kingdom) and were used at a onentration of 1. mg/ml following washing (25 x g, 1 min) and resuspension in HBSS. The numbers of surfae C3bi reeptors on both treated and untreated neutrophils were assessed by fluoresene-ativated ell sorter analysis with an PICS C ell sorter (Coulter letronis, Luton, United Kingdom) INFCT. IMMUN. by an indiret immunofluoresene monolonal antibody tehnique employing mouse anti-human C3bi and fluoresein-onjugated rabbit anti-mouse antibodies (DAKO, High Wyombe, United Kingdom) (17). Data were expressed as mean inreases in fluoresene intensity above the baseline (HBSS values) ± standard errors of the means (SMs). Gel filtration. Baterial ulture filtrates (2 ml) obtained from eah of the six strains of NTHi at 72 h (12 ml total) were mixed, lyophilized, and resuspended in 2 ml of endotoxin-free water for hromatography on a Sephadex G-5 gel olumn (81 by 1 m, medium grade; Pharmaia). lution was arried out with water at 51 ml h-1, and 5-ml frations were olleted. The eluate was monitored by determination of UV A28 and also by bioassay for neutrophil hemotaxis-inhibitory ativity at dilutions of between 1-1 and 1' in HBSS. Blue dextran, ribonulease, vitamin B12, and phenol red were used as moleular mass markers. Chromatography was performed on three separate oasions. Conseutive frations ontaining biologially ative material eluting from the olumn were pooled, yielding three peaks of ativity, and these were further frationated by passage through Detoxigel (Piere & Warriner, Chester, United Kingdom) and C18 Sep-Paks (Waters Asso., Cheshire, United Kingdom). The effiay of the Detoxigel at removing endotoxin ativity was onfirmed with the Limulus polyphemus ameboyte lysate test (-toxate kit; Sigma Poole). The biologial ativity in one of the peaks of pooled material was ompletely removed by this purifiation proedure, and the two remaining peaks of ativity obtained from endotoxin-free pooled material were referred to as peaks of partially purified neutrophil hemotaxis-inhibitory ativity. ffet of partially purified neutrophil hemotaxis-inhibitory ativity on neutrophil migration toward the hemotaxins FMLP and LTB4. Neutrophils were treated (2 min, 37 C) with two pooled frations of partially purified neutrophil hemotaxis-inhibitory ativity at dilutions of between 1-1 and 1-4 in HBSS, and the ells were allowed to migrate toward either FMLP or LTB4 in the range of 1-6 and 1-1 M, following entrifugation (25 x g, 1 min) and resuspension in HBSS. Chemial and enzymi inativation. The two pooled frations of partially purified neutrophil hemotaxis-inhibitory ativity were treated with a number of reagents. All hemials were obtained from either BDH or Sigma, and the reations were arried out at room temperature unless otherwise stated. The treatments onsisted of protease linked to Sepharose beads (Staphyloous gnseus,.1 U/ml, 37 C, 15 min), lipase linked to Sepharose beads (Candida ylindraea,.1 U/ml, 37 C, 15 min), a boiling water bath (15 min),.1 M HCl (56 C, 15 min),.1 M NaOH (56 C, 15 min), methanoli hydrogen hloride (prepared by bubbling hydrogen hloride through methanol for 2 min) for 3 h at room temperature, methanoli sodium borohydride (1,ug/ml, 3 h), methanol-aeti anhydride (3:1, vol/vol; 3 h), aqueous sodium periodate (1 mm, 3 h), and diborane in tetrahydrofuran (1 min). Controls onsisted of both buffer alone whih had been treated with reagents in a manner similar to that for the partially purified neutrophil hemotaxis-inhibitory ativity samples and samples whih had been treated with enzymes whih had been inativated by boiling. Solvents were removed under vauum, and the samples were tested for neutrophil hemotaxis-inhibitory ativity at a 12 dilution in HBSS. Letin adsorption. The two pooled frations of partially purified neutrophil hemotaxis-inhibitory ativity obtained from Sephadex G-5 were resuspended in endotoxin-free

3 VOL. 61, 1993 INHIBITION OF HUMAN NUTROPHIL MIGRATION 2421 ) 11 - (a) o lol r_ CD lo" - m (b) 1 a o los O- I Time (hours) i._ Time (hours) FIG. 1. ffets of ulture filtrates of six linial isolates of H. influenzae on neutrophil migration. (a) Baterial growth urves of six linial isolates of NTHi. (b) Chemotati (-) and hemotaxisinhibitory () ativities of ulture filtrates of six linial isolates of NTHi toward neutrophils. Chemotati ativity is expressed as a perentage (mean SM) of that aused by FMLP (1-' M) used as a standard. Maximum ativity was observed at 12.5 h and returned to basal levels by 24 h. Inhibitory ativity is expressed as a perent (mean + SM) redution of that aused by FMLP (1-9 M). Inhibitory ativity was first observed at 18 -h and had not reahed a maximum by 72 h, by whih time an appreiable drop in baterial numbers had ourred. water and passed through separate olumns (1 ml) of Tritium vulgaris wheat germ agglutinin oupled to Sepharose (Sigma). The olumn was washed with water (4 ml) followed by aqueous N-aetyl-D-gluosamine (4 ml,.5 M; Sigma). Frations were assayed at a 1-2 dilution in HBSS with N-aetyl-D-gluosamine (.5 M) as the negative ontrol. Statistial analysis. Control and test results were ompared by nonparametri statistis with the Wiloxon signed rank test. RSULTS Time ourse of appearane of ativity influening neutrophil funtions in ulture filtrates from NTHi. Six linial isolates of NTHi were ultured for up to 72 h in Tryptiase soy broth. Baterial numbers inreased logarithmially for the first 12 h, they were stationary for a further 36 h, and they thereafter delined (Fig. la). Neutrophil hemoattratant ativity was present in eah of the ulture filtrates obtained from the six 25 C. b) o._. CR F.._._ I O-' '4 1' Undiluted Dilution of 72h ulture filtrate Inubation FIG. 2. ffets of filtrate dilution and time of preinubation of neutrophils with ulture filtrates of six linial isolates of NTHi on inhibition of neutrophil migration toward FMLP. (a) Neutrophil hemotaxis-inhibitory ativity of 72-h ulture filtrates is still present after a 14-fold dilution. (b) Preinubation of neutrophils with a 1-2 dilution of 72-h ulture filtrates for 2 min resulted in maximal hemotaxis-inhibitory ativity. time (min) linial isolates of NTHi, and it rose during the log phase of baterial growth; maximal levels were ahieved at around 12.5 h, and ativity delined to basal levels by 24 h (Fig. lb). In addition to hemoattratant ativity, filtrates obtained following 12 h of ulture of the six isolates of NTHi were found to possess ativity (whih was not present at earlier time points) that inhibited human neutrophil migration toward FMLP without affeting ell viability. Chemotaxisinhibitory ativity inreased during baterial ulture and was maximal at 72 h (Fig. lb), even though the baterial ount had fallen by 2 log units (Fig. la). ffets of dilution and time of preinubation with NTHi 72-h ulture filtrates on inhibition of neutrophil migration toward FMLP; the reversibility of this effet and influene of the filtrates on neutrophil viability. Ativity was still present in filtrates diluted 14-fold, and a 1-2 dilution was used in all further experiments on the effets of 72-h ulture filtrates (Fig. 2a). The maximum redution in the response of the neutrophils to FMLP required preinubation with a 1-2 dilution of the 72-h filtrates for 15 to 2 min (Fig. 2b), and this time was used for all further inubations. The inhibitory ation of the ulture filtrate was irreversible: there was no differene in the migratory responses to FMLP for ulture filtrate-treated neutrophils whih had been washed and allowed to stand at room temperature in HBSS for 3 min (79.2% + 3.1% redution in FMLP response) or 6 min 9

4 2422 CUNDLL T AL. TABL 1. ffets of ulture filtrates (7 and 72 h) obtained from six linial isolates of NTHi on neutrophil phagoytosis and C3bi reeptor expression Result after phagoytosisa Treatment % of ells No. of C3bi reeptor ontaining beads/ expression beads ell HBSS (ontrol) 59.9 ± ± ±.1 7-h ulture filtrate O 9.5 ± ± o.3 72-h ulture filtrate ± ±.4 FMLP (1-3 M) ± ±.8 a Perentages of ells ontaining beads and numbers of beads per ell were determined at 3 min. Data are expressed as means ± SMs of values obtained with the six linial isolates. b xpressed as ratio of inrease in fluoresene intensity above the baseline (HBSS values). Data are expressed as means ± SMs of the inrease produed with the six linial isolates. C P <.1 versus ontrol by Wiloxon signed rank test. (8.4% ± 2.3%) and those for treated neutrophils tested immediately (82.2% ± 2.7%). Inubation with ulture filtrates for up to 9 min did not affet the viability of the neutrophils as assessed by trypan blue exlusion (all ells were >96% viable) or latate dehydrogenase release (<5% of total latate dehydrogenase was released by 9 min). ffets of 7- and 72-h NTHi ulture filtrates on neutrophil hemotaxis and hemokinesis. Seven-hour ulture filtrates obtained from the six isolates of NTHi ontained signifiant levels of hemoattratant (mean perentage of FMLP migration = 45.9% ± 1.%) ompared with those in ontrols (2.2% ±.9%) at all onentrations (dilutions of 1-1 to 1-'; P <.1). By hekerboard analysis, this ativity was found to be primarily hemokineti, with some assoiated hemotati ativity. In ontrast, 72-h ulture filtrates did not produe hemotaxis or hemokinesis at any onentration studied (mean perentage FMLP migration = 3.1% ± 1.2%). ffets of 7- and 72-h NTHi ulture filtrates on neutrophil phagoytosis and C3bi reeptor expression. Following inubation of neutrophils with the 7-h filtrates, there was a signifiant (P <.1) inrease in their phagoyti apaity and a 22.8% inrease in their C3bi surfae reeptors (Table 1). This is in agreement with the presene of a hemoattratant in these filtrates leading to ellular ativation (15). The 72-h filtrates had no effet on neutrophil phagoytosis, although a small but signifiant derease in C3bi reeptor expression was observed, indiating that the inhibitory effets on migration did not appear to be due to ellular ativation (11.6% ± 3.1%; P <.1) (Table 1). The biologial signifiane of the small derease in neutrophil C3bi reeptors is not lear. Partial purifiation of ativity inhibiting FMLP-indued neutrophil migration (neutrophil hemotaxis inhibitory ativity) from 72-h NTHi ulture filtrates. A lyophilized sample of the pooled 72-h ulture filtrates was hromatographed on Sephadex G-5, and three peaks of hemotati inhibitory ativity were observed (Fig. 3). The ativity assoiated with peak I (V/VO = 1.1) was ompletely removed by passage through Detoxigel, suggesting that it was endotoxin, whih is known to affet neutrophil funtion. This is onsistent with previous studies on endotoxin and neutrophil priming (9). In ontrast, Detoxigel treatment of peaks II (V/VO = 2.8) and III (V/VO = 3.5) did not remove the inhibitory ativity. Inhibitory ativity present in peaks II and III was retained on a C18 Sep-Pak olumn and ould be eluted in methanol (frations SPIIb and SPIII). Frations SPIIb and SPIII redued the +-f I Ē s1 9'- 75. rc 6. I._ - 45 C 3 r- 15 4) INFCT. IMMUN. Ribonulease A Blue dextran V a Fration number (5 ml) FIG. 3. Gel filtration on Sephadex G-5 of pooled 72-h ulture filtrates from six linial isolates of NTHi resulting in three peaks of hemotaxis-inhibitory ativity. Fration I is assoiated with endotoxin. Blue dextran (2 x 13 kda), ribonulease (13.7 kda), vitamin B12 (1.355 kda), and phenol red (.354 kda) were used as moleular mass markers. Optial density at 28 nm (ODm,) is shown as a broken line. hemotati responses of neutrophils to both FMLP and LTB4 (1-6 to 1-1o M) at all dilutions tested (1-1 to 1-4), with maximal effets being observed at a dilution between 1-1 and 1-2 in HBSS (data shown for 1-2 dilution in HBSS in Fig. 4). Peak II also ontained substanes whih did not bind to the solid-phase olumn (fration SPIIa). It is not known whether this represents weak binding of the major bioative speies to the Sep-Pak or there are other, more polar substanes present. This fration (SPIIa) was not examined further. Biohemial analysis of partially purified neutrophil hemotaxis-inhibitory ativity (after Sephadex G-5, Detoxigel, and C18 Sep-Pak). The ative speies present in frations SPIIb and SPIII ould not be extrated into hloroform, suggesting that the neutrophil hemotaxis-inhibitory substanes were not assoiated with simple lipids. Boiling, aid and base treatment, and borohydride redution had no effet on the ativity of either fration SPIIb or fration SPIII (Table 2). The ativities of both frations were destroyed (>9%) by proteolyti digestion, indiating the presene of a peptide or protein, by treatment with sodium periodate (whih attaks is glyols suh as sugars), and by diborane redution (whih attaks double bonds) (Table 2). Partial inativation (>55%) was ahieved by lipase treatment (whih attaks lipids) and aetylation (whih attaks hydroxyl and amine groups) (Table 2). These data are onsistent with the bioative fators ontaining peptides or proteins, lipids, sugars, double bonds, and hydroxyl, arboxyl, and amine groups. Ativity was ompletely retained on a wheat germ agglutinin olumn and ould be eluted with N-aetyl-D-gluosamine. These data are onsistent with the inhibitory substanes being N-aetyl-Dgluosamine-ontaining glyopeptides, perhaps linked through an ayl group to a lipid. The higher moleular mass of fration SPII (as determined by gel filtration) and the redued reovery of ativity on a C18 Sep-Pak would be onsistent with inreased glyosylation

5 VOL. 61, 1993 INHIBITION OF HUMAN NUTROPHIL MIGRATION 2423 to ) 9-J L-j.61. a to m.- o a a) b) 1 - o if-7 L-,( lo-,, FMLP (M) TABL 2. Biohemial analysis of partially purified neutrophil hemotaxis-inhibitory ativity from 72-h ulture filtrates of linial isolates of NTHi Treatment or substane used in Result" (%) for fration: treatment SPIIb SPIII None Freeze-thawing ph stabilization (aid) 1.4 ±.9.5 ±.4 ph stabilization (alkali).3 ± ± 2.4 Boiling 4.2 ± 1..3 ±.1 Protease 96. t ± 3.7 Lipase 67.3 ± ± 2.9 Sodium borohydride ± 1.5 Sodium periodate 93.4 ± ± 2.3 Pyridine-aeti anhydride 63.3 ± ± 1.7 Methanol-aeti anhydride 88.9 ± Methanol-hydrohlori aid 22.1 ± ± 1.7 Diborane 83.2 ± t 1.3 / Ativity was obtained by Detoxigel and C18 Sep-Pak frationation of Sephadex G-5 gel filtration peaks II and III. Values given in the table are perent falls in the levels of neutrophil hemotaxis inhibitory ativity from untreated values. Ativity is the perent redution in migratory response to FMLP (1-' M). Bakground migration has been subtrated from all values shown, whih are means SMs (n = 3). 25 desribed in this study are not diretly ytotoxi and do not C inrease neutrophil movement (either hemotati or hemokineti), phagoytosis, or C3bi reeptor expression, sug- - - gesting that this is not simply a generalized effet on the r..,. neutrophil ell wall that produes neutrophil ellular ativa tion. The dose-response urves to both hemotaxins are LTB4 (M) flattened by the fators, whih suggests that the effets on FIG. 4. Frations SPIlb and SPIII (after Sephadex G-5, neutrophil migration toward the hemotati fators FMLP Deto igel, and a C18 ' Sep-Pak) at a 1-2 dilution in HBSS suppressed the and LTB4 are not related to an alteration in reeptor number. hemotati dose-response urve to both FMLP (a) and LTB4 (b). A similar flattening of the dose-response urve to FMLP also Data are expresssed as means + SMs (n = 6) for the hemotaxin ours in response to treatment of neutrophils with pertussis ontrol (*), fration SPIIb (), and fration SPIII (). toxin, whih similarly produes its effets without loss of ellular viability (1). Pertussis toxin is known to inhibit neutrophil hemotaxis and enzyme seretion and is believed DISCUSSION to produe these effets by bloking the a subunit of Gi, the inhibitory GTP-binding regulatory protein. Polymorph)nulear leukoytes are reruited to sites of The inhibitory fators have not yet been haraterized. infetion in re-sponse to the prodution of endogenous and Gel filtration of a pooled 72-h filtrate yielded two major baterium-derived hemotaxins (12). These ells play a key peaks of low-moleular-mass (<15-kDa) hemotaxis-inhibifense against baterial infetions of muosal tory ativity whih were not assoiated with endotoxin. role in the de surfaes (5, 1.3). Several pathogeni speies, inluding B. Some of the ativity assoiated with one of the peaks (peak pertussis (1), L. midadei (7), and P. aeruginosa (2, 1), II) was not bound to a C18 Sep-Pak olumn, and it is not lear release substzanes whih either are diretly ytotoxi to whether this represents weak binding of the major bioative neutrophils or at through inhibiting signal transdution, and speies to the Sep-Pak or there are other, more polar this may be ain important fator in their survival and prolif- substanes present. This is urrently being investigated. The eration in the lung. studies presented in this paper have onentrated on the We have si iown that six linial isolates of the ommon ativity bound to C18 Sep-Pak olumns whih ould be respiratory palthogen NTHi produe fators whih adversely eluted by methanol (frations SPIIb and SPIII). Chemial affet neutrop)hil funtion. As do many other respiratory and enzymi inativation studies of these two frations are trat bateria, NTHi generated neutrophil hemoattratants onsistent with the presene of N-aetyl-D-gluosamineand stationary phases ( to 18 h) of growth ontaining glyopeptides, perhaps linked to lipids. As bio- during the log (13). After 12 h of ulture, this attratant ativity diminished ativity was not retained on the endotoxin-affinity matrix, and was repllaed by fators whih inhibited neutrophil Detoxigel, it is unlikely that these fators ontain lipid A. migration tow'ard two unrelated hemotaxins (FMLP and The presene of N-aetyl-D-gluosamine residues suggests LTB4). Inhibiitory ativity inreased with time and was that the neutrophil hemotaxis-inhibitory ativity deteted maximal at 72 h, even though the baterial viable ount had may derive from the baterial ell wall. Several gram- effet on migration was not reversible, negative bateria, inluding sherihia oli, Salmonella fallen. This inlhibitory although the Sells remained viable for up to 9 min. typhimurium, and Neissena gonorrhoeae, release peptinism of ation of the Haemophilus-derived doglyans during ell division (8, 14), and some fragments The meha: inhibitory fattor(s) I I is not known. The Haemophilus fators have moleular masses similar to those of frations SPIIb

6 2424 CUNDLL T AL. and SPIII (14). Although the majority of the peptidoglyan is reinorporated by the bateria, in the ase of. oli, up to 8% per generation is lost (8), and for N. gonorrhoeae, as muh as 35% per generation is lost (14). Peptidoglyans have been previously shown to have numerous ativities, inluding inhibition of marophage hemotaxis, ativation of omplement (13), and the indution of pulmonary inflammation in a rabbit model (18). Large fragments (35 to 4 kda) of peptidoglyan (3 to 45 kda) may also stimulate human mononulear ells to produe fators whih inhibit neutrophil hemotaxis toward FMLP without affeting either ellular viability or phagoytosis (6), although their diret effets on human neutrophil migration remain unknown. NTHi organisms very ommonly olonize the lower respiratory trats of patients with hroni airway diseases, and they are assoiated with infetive exaerbations. Infetions of the lower airways are assoiated with neutrophil infiltration, whih results from the interation of neutrophils with a number of loally derived endogenous and baterially derived hemotati fators, whih in turn leads to their ativation. Host-derived hemotati fators inlude the omplement omponent CSa, LTB4, platelet-ativating fator, and leukoyte-derived ytokines (13). Our data show that while some produts released earlier in the growth yle are hemoattratants, other produts released later inhibit hemotaxis. If these inhibitory fators are released into the miroenivironment of the bronhial muosa, they might interfere with baterial learane by neutrophils. This might help to explain why these bateria are not eradiated in hroni bronhial infetions despite there being a normal neutrophil response (16). ACKNOWLDGMNTS We thank Lilly Industries for finanial support. We thank Christopher Haslett, Department of Mediine, University of dinburgh, for his advie. RFRNCS 1. Beker,. L., J. C. Kermode, P. H. Naahe, R. Yassin, M. L. Marsh, J. J. Munoz, and R. I. Sha'afi The inhibition of neutrophil granule enzyme generation and hemotaxis by pertussis toxin. J. Cell Biol. 1: Bishop, M. B., A. L. Balth, L. A. Hill, R. P. Smith, F. Lutz, and M. Pollak The effet of Pseudomonas aeruginosa ytotoxin and toxin A on polymorphonulear leuoytes. J. Med. Mirobiol. 24: Borgeat, P., and B. Samuelsson Metabolism of arahidoni aid in polymorphonulear leukoytes. ffet of alium INFCT. IMMUN. ionophore A Pro. Natl. Aad. Si. USA 76: Cline, M. J., and R. I. Lehrer Phagoytosis by human monoytes. Blood 32: Currie, D. C., A. M. Peters, P. George, et al llilndiumlabelled granuloyte aumulation in respiratory trat of patients with bronhietasis. Lanet i: Donabedian, H Human mononulear ells exposed to staphylooi rapidly produe an inhibitor of neutrophil hemotaxis. J. Infet. Dis. 152: Donowitz, G. R., I. Reardon, J. Dowling, L. Rubin, and D. Foht Ingestion of Legionella midadei inhibits human neutrophil funtion. Infet. Immun. 58: Doyle, R. J., J. Chaloupka, and V. Vinter Turnover of ell walls in miroorganisms. Mirobiol. Rev. 52: Guthrie, L. A., L. C. MPhail, P. M. Hendon, and R. B. Johnston Priming of neutrophils for enhaned release of oxygen metabolites by baterial lipopolysaharide. J. xp. Med. 16: Kharazmi, A., G. Doring, N. Hoiby, and N. H. Valerius Interation of Pseudomonas aeruginosa alkaline protease and elastase with human polymorphonulear leuoytes in vitro. Infet. Immun. 43: Lee, T. H., L. Nagy, T. Nagakura, M. J. Walport, and A. B. Kay Identifiation and partial haraterization of an exerise-indued neutrophil hemotati fator in bronhial asthma. J. Clin. Invest. 69: Maleh, H. I., and J. I. Gallin Current onepts: immunology. Neutrophils in human diseases. N. ngl. J. Med. 317: Ras, G., R. Wilson, G. Taylor, and P. Cole ffet of baterial produts on neutrophil migration in vitro. Thorax 45: Rosenthal, R. S Release of soluble peptidoglyan from growing gonooi: hexaminidase and amidase ativities. Infet. Immun. 24: Sheinman, B. D., J. L. Devalia, S. J. Crook, and R. J. Davies Synthesis of histamine by Haemophilus influenzae. Br. Med. J. 292: Stokley, R. A., J. Shaw, S. L. Hill, and D. Burnett Neutrophil hemotaxis in bronhietasis: a study of peripheral ells and lung seretions. Clin. Si. 74: Tetteroo, P. A., M. J.. Bos, F. J. Visser, and A.. G. K. R. von dem Borne Neutrophil ativation deteted by monolonal antibodies. J. Immunol. 136: Tuomanen,., R. Rih, and. Zak Indution of pulmonary inflammation by omponents of the pneumooal ell surfae. Am. Rev. Respir. Dis. 135: Turk, D. C The pathogeniity of Haemophilus influenzae. J. Med. Mirobiol. 18: Zigmond, S. H., and J. G. Hirsh Leuoyte loomotion and hemotaxis: new methods for evaluation of a ell-derived hemotati fator. J. xp. Med. 137: