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Blood, 1 February 2004, Vol. 103, No. 3, pp. 1105-1113. Prepublished online as a Blood First Edition Paper on September 25, 2003; DOI 10.1182/blood-2003-08-2780.
PHAGOCYTES Unique CD18 mutations involving a deletion in the extracellular stalk region and a major truncation of the cytoplasmic domain in a patient with leukocyte adhesion deficiency type 1From the Department of Pediatrics, Section of Leukocyte Biology, Baylor College of Medicine, Houston, TX; and Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, OH.
Two novel CD18 mutations were identified in a patient who was a compound heterozygote with type 1 leukocyte adhesion deficiency and whose phenotype was typical except that he exhibited hypertrophic scarring. A deletion of 36 nucleotides in exon 12 (1622del36) predicted the net loss of 12 amino acid (aa) residues in the third cysteine-rich repeat of the extracellular stalk region (mut-1). A nonsense mutation in exon 15 (2200G>T), predicted a 36-aa truncation of the cytoplasmic domain (mut-2). Lymphocyte function-associated antigen 1 (LFA-1) and macrophage antigen-1 (Mac-1) containing the mut-1  2 subunit were expressed at very low levels compared with wild-type (wt) 2. Mac-1 and LFA-1 expression with the mut-2 2 subunit were equivalent to results with wt 2. Binding function of Mac-1 with mut-2 2 was equivalent to that with wt 2. However, binding function of LFA-1 with the mut-2 2 subunit was reduced by 50% versus wt 2. It was concluded that (1) the portion of the CD18 stalk region deleted in mut-1 is critical for 2 integrin heterodimer expression but the portion of the cytoplasmic domain truncated in mut-2 is not; and (2) the mut-2 cytoplasmic domain truncation impairs binding function of LFA-1 but not of Mac-1. Studies with the patient's neutrophils (PMNs) were consistent with functional impairment of LFA-1 but not of Mac-1. (Blood. 2004;103:1105-1113)
Important insights related to the molecular mechanisms that determine 2 integrin structure and function have arisen from the study of naturally occurring mutations in the common 2 subunit of this integrin family.1,2 A range of mutations in the CD18 gene have been identified in patients with leukocyte adhesion deficiency type 1 (LAD-1), including deletions, truncations, substitutions, frame shifts, and intronic mutations that result in abnormalities of the CD18 protein ranging from absence of protein product, deficient alpha-beta subunit association, and reduced protein size.1,3-5 All such mutations have resulted in diminished expression of the CD18 integrins on leukocytes, and some mutations also have caused integrin dysfunction.1,3-5 Patients typically have recurrent bacterial or fungal infections of skin and mucous membranes, impaired mobilization of leukocytes to sites of infection, severe gingivitis/periodontitis with permanent loss of dentition, and atrophic scarring of wounds.1,2,6 Laboratory features include a pronounced leukocytosis in the absence of infection, reduced expression of all members of the CD18 or 2 integrin family on circulating leukocytes, and diminished CD18-dependent leukocyte functions, including cell adhesion, chemotaxis, transendothelial migration, and oxidative burst activation in response to iC3b-coated particles.1,2,6 We have identified an adult male patient with LAD-1 whose clinical features resemble those of the moderate LAD-1 phenotype, except that his wounds heal with hypertrophic rather than atrophic scarring. His levels of CD18 integrin expression are higher than those of most other reported patients with the moderate phenotype of LAD-1, but the CD18-dependent functions of his leukocytes were similar to those of other moderate phenotype individuals.1,7 He was found to be a compound heterozygote with 2 novel mutations, one of special interest in that it involves a major truncation of the cytoplasmic domain of CD18. Studies of COS-7 cells transfected with his mutant CD18 alleles and of his native leukocytes revealed distinct influences of his mutations on integrin expression and function. A quantitative relationship was established between the amount of macrophage antigen-1 (Mac-1) present at the neutrophil (PMN) surface, including translocated Mac-1, and the capacity of the PMNs for Mac-1-mediated adhesion induced by a subsequent optimal stimulus.
Subject and controls The patient is a 35-year-old man who was reported previously by Shurin et al8 in 1979 as having a disorder of PMN chemotaxis presumed to be caused by the overgrowth in the oral cavity of Capnocytophaga, sonicates of which contained a dialyzable inhibitor of PMN chemotaxis. His clinical course to that time was characterized by early severe periodontal disease with loss of alveolar bone, persistent neutrophilia, and recurrent boils containing clear fluid. Over the last 20 years he has been generally healthy, except for hypertrophic scarring of skin cuts and abrasions and 2 episodes of acute abdominal pain associated with terminal ileitis that resolved with intravenous antimicrobial therapy. He has had permanent dentures for over 25 years, and he has been relatively free of gingival symptoms. A more recent orolabial infection due to Candida albicans and peripheral blood PMN counts of more than 20 x 109/L (20 000/µL) for several weeks after successful treatment prompted a reevaluation of his PMN function. Because many aspects of this patient's clinical course were similar to those previously described for patients with the moderate phenotype of LAD-1,9 a disorder not yet characterized when he was reported originally, subsequent laboratory evaluation focused on that disorder. Both parents and a 38-year-old sister, all in good health, were also studied. Healthy adult volunteers provided blood leukocytes for use as controls. Flow cytometric analysis of PMNs from the patient confirmed abnormally low expression levels of CD18 integrins. Monoclonal antibodies and other reagents
Monoclonal antibodies (mAbs) against CD11a included TS1/22 (provided by Dr Timothy Springer, Boston, MA) and R3.1 (provided by Robert Rothlein, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT); mAbs against CD11b included M1/70 (hybridoma clone purchased from American Type Culture Collection [ATCC], Manassas, VA), 60.1 (provided by John Harlan, Seattle, WA), and anti-Leu15 (Becton Dickinson, Mountain View, CA); mAbs against CD11c included anti-LeuM5 (Becton Dickinson); mAbs against CD18 included R15.7 (R. Rothlein), TS1/18 (T. Springer), and MHM23 (ATCC). Additional mAbs included anti-CD35 (CR1; clone 3D9, from Dr Melvin Berger, Cleveland, OH), anti-CD32 (Fc Isolation of PMNs and mononuclear cells from peripheral blood PMNs and mononuclear cells were isolated from peripheral blood as previously described,10 with minor modifications, using dextran sedimentation of erythrocytes and centrifugation of cells from the top leukocyte layer over a Ficoll-Hypaque cushion. Both the purity and viability of the pelleted PMNs prepared by this method were uniformly 95% or higher. For some studies, residual red blood cells in the pellet were removed by hypotonic lysis. Mononuclear cell fractions from the interphase layer were used to prepare immortalized B-lymphocyte lines by transformation with Epstein-Barr virus11 (EBV) and as a source of cellular RNA. cDNA amplification RNA was isolated from mononuclear leukocytes using TRIZOL Reagent (Invitrogen Life Technologies, Carlsbad, CA) and reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen), followed by amplification of the full-length CD18 cDNA as 4 overlapping fragments using the primers listed in Table 1. The polymerase chain reaction (PCR) conditions were as follows: one cycle at 95°C for 5 minutes followed by 35 cycles of 1 minute at 95°C, 1 minute at 58°C, 1 minute at 72°C, and a final step of 10 minutes at 72°C. The PCR products were electrophoresed on 1% agarose gels and purified with the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). The purified fragments were sequenced by the Core Laboratory Services of the Children's Health Research Center (Department of Pediatrics, Baylor College of Medicine) using the 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Subcloning and transformation Full-length CD18 cDNA was obtained by ligating together the above PCR products that showed sequence integrity and then subcloned into the shuttle vector pcDNA3.1 (Invitrogen). The restriction sites for ligation were as follows: BglI (T674/T675) for fragments 1 and 2; EcoRI (G1069/A1070) for fragments 2 and 3; and AatII (T1748/C1749) for fragments 3 and 4. The cDNAs encoding CD11a and CD11b, subcloned in the expression vectors pAPRM8 and pCDM8, respectively, were generous gifts from Dr T. Springer.
Bacteria of the electrocompetent Escherichia coli strain ElectroMAX DH5 Plasmid preparation and cotransfection of COS-7 cells Random bacterial colonies were selected and grown overnight at 37°C for small-scale plasmid preparation (QIAGEN Plasmid Mini Kit). After confirming the sequence integrity of CD18 cDNA inserts, large-scale plasmid preparation was performed using either the QIAGEN Plasmid Midi or Maxi Kit. Transfection of COS-7 cells was carried out using Lipofectamine 2000 (Invitrogen) and performed according to the manufacturer's recommendations, using 5 µg of CD11a or CD11b cDNA and 5 µg of CD18 cDNA for each 25 cm2 flask of COS-7 cells at about 90% confluence. The cells were incubated for an additional 48 hours to allow expression of the integrin constructs. Immunofluorescence flow cytometry for expression of CD11/CD18 molecules on COS-7 cells or blood leukocytes Staining and analysis for direct immunofluorescence were performed for COS-7 cells as previously described.13 Results were corrected for background fluorescence obtained with nonbinding isotype-matched mAbs. Leukocytes in whole blood also were stained with directly conjugated mAbs and analyzed in a FACScan (Becton Dickinson) flow cytometer, with gating of cells by 90° light scatter for granularity and forward light scatter for cell size to distinguish PMN, lymphocyte, or monocyte populations. For some studies, the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLF) was added at concentrations up to 10 nM for 20 minutes at 37°C to partially or fully up-regulate PMN surface expression of CD11b/CD18 prior to staining. Results were corrected for background fluorescence. Estimation of the number of Mac-1 surface sites on PMNs was achieved using a calibrated set of antibody-bound bead standards, as previously described.14 For blood lymphocyte populations, 2-color fluorescence was used, with gating achieved using a fluorescein isothiocyanate (FITC)-conjugated population marker (anti-CD3, anti-CD4, or anti-CD8) and CD18 or a phycoerythrin (PE)-conjugated mAb for CD11a expression. Northern transfer and hybridization Total RNA was extracted from transfected COS-7 cells using TRIZOL Reagent (Invitrogen). RNA (10 µg) was loaded onto agarose gels containing formaldehyde, electrophoresed, and transferred to nylon membranes (GeneScreen Plus; PerkinElmer Life Sciences, Boston, MA), which were then baked at 80°C for 1 hour in a vacuum oven. CD18 fragment 2 cDNA (see Table 1) was used as a probe for CD18 message. The labeled probe was prepared with the Prime-It II Random Primer Labeling Kit (Stratagene Cloning Systems, La Jolla, CA) according to the manufacturer's protocol. After a 30-minute prehybridization with ULTRAhyb (Ambion, Austin, TX), overnight hybridization with the cDNA probe (25 ng) was carried out at 48°C. Abundance of hybridized mRNA was evaluated by autoradiography. Binding to solid-phase ICAM-1 by cotransfected COS-7 cells
As described previously13,15 with modifications, flat-bottomed wells of 96-well microtiter plates (Immulon IV; Dynatech, Alexandria, VA) were coated overnight at 4°C with 3.0 µg recombinant human intercellular adhesion molecule-1 (ICAM-1), washed, and blocked with 5% bovine serum albumin (BSA) for 1 hour at 37°C. After activation of COS-7 cells by treatment with the activating anti-CD18 mAb KIM185, COS-7 cells (105 in 0.1 mL) were added in triplicate to each well and allowed to settle onto the surface of the well for 15 minutes at 37°C. Nonadherent cells were carefully removed by 3 washes with phosphate-buffered saline (PBS). The number of cells retained at the bottom of each well was counted microscopically. Additional pretreatment of COS-7 cells with mAb R3.1 against LFA-1 ( Rosetting of COS-7 cells with iC3b-coated sheep erythrocytes (iC3b-SRBCs)
An adaptation of previously described methods was employed.16-18 Briefly, a 10% vol/vol suspension (0.3 mL) of SRBCs (ICN Biomedicals, Aurora, OH) was incubated for 60 minutes at 25°C with 30 µg rabbit anti-SRBC immunoglobulin M (IgM; ICN Biomedicals). The IgM-coated SRBCs were washed, resuspended in C5-depleted human serum (Sigma-Aldrich, Dallas, TX) diluted to 25% in PBS, and incubated for 30 minutes at 37°C to permit opsonization of the IgM-SRBCs with iC3b. After thorough washing, approximately 5 x 107 opsonized SRBCs were added to 105 COS-7 cells in a final volume of 150 µL. These mixtures were centrifuged gently (150g for 3 minutes) to form a loose pellet then incubated at 37°C for 30 minutes. The pellets were resuspended and each suspension was examined by light microscopy for rosette formation, defined as 3 or more RBCs bound to one COS-7 cell (rosettes rarely contained fewer than 10 RBCs). An mAb against Assays for PMN adhesion and transendothelial migration Suspensions of PMNs were injected into static adhesion chambers19,20 and allowed to settle onto a coverslip coated with either keyhole limpet hemocyanin (KLH) or a monolayer of human umbilical vein endothelial cells (HUVECs), prepared as previously described.20 After incubation at 37°C for 500 seconds, the chamber was inverted and nonadherent PMNs were allowed to settle onto an uncoated glass coverslip on the opposite side of the chamber. The percentage of PMNs that remained adherent to the substrate was determined by phase contrast microscopy. Of PMNs that were adherent to a HUVEC monolayer, those that traversed the endothelium were then distinguished by their flatter, "phase-dark" appearance, in contrast to the "phase-bright" appearance of PMNs that remained at the lumenal surface of the monolayer.20 PMN aggregation in response to 1.0 µM fMLF was measured by the flow cytometric method of Rochon and Frojmovic.21 In vivo migration of PMNs into skin suction blister fluid at successive time intervals was measured by adapting a previously described technique.22 Chemiluminescence assay for PMN oxidative burst in response to opsonized zymosan particles PMNs (106 cells in a final volume of 1.0 mL) were mixed in a cuvette at 37°C with 1 mg serum-opsonized zymosan particles, prepared as previously described,6,7 and 10-5 M lucigenin (bis-N-methyl acridinium nitrate).23 At 5-minute intervals, the light that was emitted upon the reduction of lucigenin was measured in a temperature-controlled luminometer (model 1201; LKB-Wallac, Turku, Finland) and expressed in millivolts as a measure of PMN respiratory burst activation.23,24 Mac-1 dependence was confirmed by blocking the receptor on PMNs with mAb 60.1. Phorbol myristate acetate (PMA) was used as a nonparticulate stimulus to confirm intrinsically normal respiratory burst activity.
Analysis of patient's CD18 sequence data A mutation was identified in the patient's CD18 sequence, characterized by a deletion of the last 36 nucleotides of exon 12 (1622del36; "A" of initiating ATG codon = 1). This deletion predicts a net loss of 12 amino acid (aa) residues in the third cysteine-rich repeat of the CD18 extracellular stalk region, with loss of 13 original aa residues and the insertion of a new tryptophan residue due to the formation of a new codon by nucleotides at the margins of deletion (C541_G553delinsW).25 This mutation, referred to below as "mut-1," is diagrammed in Figure 1A.
Returning to the patient's mononuclear cell RNA, additional PCR reactions using new primers closely flanking the above mutation yielded 2 distinct, equally intense product bands (data not shown), suggesting that the patient was heterozygous for this deletion. A second mutation was identified as a nonsense mutation in exon 15 (2200G>T), predicting a 36-aa truncation of the cytoplasmic domain of CD18 (E734X). This mutation, referred to below as "mut-2," is diagrammed in Figure 1B. The CD18 precursor, with designated locations of mut-1 and mut-2, is depicted in Figure 1C.
Cell surface expression of LFA-1 and Mac-1 in COS-7 cells cotransfected with the wild-type (wt)
COS-7 cells cotransfected as described were analyzed for LFA-1 or Mac-1 expression by immunofluorescence flow cytometry. Fluorescence histograms for a representative set of transfections are shown in Figure 2. A composite of 4 similar experiments is represented in Figure 2B. Levels of expression of Mac-1 and LFA-1 were profoundly reduced for cells transfected with mut-1
Adhesion of LFA-1-transfected COS-7 cells with wt, mut-1, and mut-2
As shown in Figure 3A, adhesion to ICAM-1 in microtiter wells was profoundly reduced for
Rosetting of Mac-1-transfected COS-7 cells with iC3b-opsonized sheep erythrocytes
As shown in Figure 3B, Surface expression of CD11/CD18 molecules on leukocytes from patient and family members compared with healthy controls
The average levels of CD18 integrin expression on the patient's leukocyte populations was 21% of healthy adult levels (Table 2). On unstimulated PMNs, CD11a was expressed at about 50% of normal levels and CD11b was approximately 17% of normal levels. A translocatable granular pool of CD11b in the patient's PMNs can be inferred from the 9-fold increase in CD11b surface levels after fMLF stimulation. CD11a and CD18 expression on the patient's peripheral blood lymphocytes ranged from 15% to 25% of control levels. Additional data not shown in the table include the following: (1) normal expression of L-selectin (CD62a), CR1 (CD35), and Fc
Adhesive functions of the patient's PMNs and the role of translocated Mac-1 The patient's PMNs exhibited marked reductions in adherence to KLH-coated glass and homotypic aggregation, the former a Mac-1-mediated process (Figure 4A and 4B, respectively). The patient's PMN oxidative burst in response to serum-opsonized zymosan was moderately reduced, with peak chemiluminescence values at 57% of control values (Figure 4C), and the response by both patient and control PMNs was reduced to baseline by the anti-Mac-1 mAb 60.1. Stimulation with PMA revealed an intrinsically normal respiratory burst mechanism (not shown).
Since previous studies have shown that newly translocated surface Mac-1 can also be activated for adhesion, we determined the extent of Mac-1-dependent adhesion by the patient's PMNs after graded chemoattractant-induced translocation of Mac-1. This analysis requires 2 sequential stimuli, one to mobilize Mac-1 to the cell surface followed by a second to activate adhesion via newly translocated Mac-1.14 The patient's PMNs were first exposed to fMLF at suboptimal concentrations from 0.1 to 6.0 nM. Half of the cells from each condition were removed and stained to quantitate Mac-1 surface expression, and the other half were exposed to fMLF a second time at 10.0 nM and assayed for PMN adhesion to KLH-coated glass. As shown in Figure 5, with increasing concentrations of fMLF, there was a progressive increase both in surface expression of CD11b on the PMNs after the initial stimulus (Figure 5A) and in adherence by PMNs after the second fMLF stimulation (Figure 5B). When the relation between Mac-1 expression and percent PMN adherence for the patient's cells was analyzed by linear regression (Figure 5C), there was a strong direct relationship between adhesion after the second stimulus and the number of Mac-1 sites on the cell surface prior to the second stimulus (regression coefficient [R] = 0.971). Thus, the patient's PMN granular pool of Mac-1 can be activated for cell adhesion after translocation to the cell surface and that Mac-1 expressed on the patient's PMNs can bind to an appropriate ligand as effectively as wild-type Mac-1, consistent with the above rosetting data with COS-7 cells expressing Mac-1 with the patient's mut-2
Adhesion by the patient's PMNs to activated HUVEC monolayers, transendothelial migration in vitro, and the effects of blocking mAbs As shown in Figure 6, the adhesion of the patient's PMNs to cytokine-activated endothelium was markedly higher than to protein-coated glass (Figure 4A) but transmigration was low. This is consistent with earlier data indicating that transendothelial migration is largely CD11/CD18 dependent, while adhesion, per se, can be mediated by other molecules as well, including PMNs and endothelial selectins.20,26 When E-selectin on endothelial cells was blocked with mAb CL2, adhesion by the patient's PMNs to the HUVECs was reduced by about 50% (not shown), confirming the participation of selectins in this process.
As previously reported for control PMNs,20 blocking CD18 almost completely inhibited transendothelial migration for both patient and control PMNs (Figure 7). For control PMNs, blocking CD11b had a relatively small inhibitory effect compared with blocking CD11a, also consistent with previous data.20 However, transmigration of the patient's cells was blocked by mAbs to either CD11a or CD11b as effectively as by anti-CD18. It seems likely that CD11a and CD11b expression is sufficiently low on the patient's cells that blocking either one abrogates transmigration. Accumulation of the patient's PMNs in skin suction blister fluid by 6 hours after blister induction was approximately 30% of adult control values (data not shown), similar to the above comparisons for transmigration.
One of the current patient's CD18 mutations (mut-1) was a deletion of 36 nucleotides in exon 12, predicting a deletion of 13 aa residues and the insertion of a tryptophan residue (C541_G553delinsW), a net loss of 12 aa residues. This deletion in the third cysteine-rich repeat domain of the extracellular stalk region eliminates 2 cysteine residues at positions 541 and 549 that participate in disulfide bonds important for CD18 tertiary structure.27 The extracellular stalk region of CD18 has been shown to be important for effective integrin heterodimer formation, and the cysteine-rich repeats near the carboxy-terminal end of the stalk region contain epitopes that define conformational changes critical for activation of alpha subunit binding.27,28 Two previous studies concluded that the "major cysteine-rich region" is not required for Mac-1 or LFA-1 surface expression.28,29 However, the patient's mut-1 deletion results in very low levels of surface expression of both LFA-1 and Mac-1, suggesting that specific deletions in this region can alter the CD18 structure sufficiently to cause a profound impairment of integrin heterodimer formation and expression, presumably resulting in intracellular degradation of most monomeric subunits that do not form heterodimers, as reviewed previously.1 Northern analysis of CD18 mRNA in transfected COS cells revealed that the low surface expression of Mac-1 and LFA-1 with mut-1 was not accounted for by reduced transcription. PCR studies with RNA from the patient's mononuclear cells and primers closely flanking the mut-1 deletion initially revealed the patient's status as a compound heterozygote, based on the presence of 2 distinct, equally intense bands from the same reaction and, while not quantitatively definitive, suggested no selective advantage for either mut-1 or the alternate allele, mut-2, at the RNA level. The very low ligand binding by LFA-1- and Mac-1-transfected COS-7 cells that contained the mut-1 2 subunit were in keeping with the low levels of cell surface expression.
The patient's second mutation (mut-2) was a nonsense mutation in exon 15, predicting a 36-aa truncation of the CD18 cytoplasmic domain (E734X). To our knowledge, this is the first reported CD18 mutation associated with LAD-1 that specifically involves the cytoplasmic domain. Progressive truncations of the CD18 cytoplasmic domain produced by site-directed mutagenesis have not appeared to have significant effects on CD18 integrin cell surface expression,13 and our studies showing equivalent expression of Mac-1 or LFA-1 in cotransfected COS-7 cells with the patient's mut-2 compared with wt
The CD18 cytoplasmic domain engages in important interactions with the cytoskeleton and appears to regulate several important aspects of
COS-7 cells expressing LFA-1 with the patient's mut-2
That Mac-1-dependent binding (rosetting) was equivalent for COS-7 cells cotransfected with mut-2 versus wt PMN adhesion after the second stimulus in the above model of sequential fMLF stimulation was directly proportional to the number of Mac-1 sites present on the cell surface after the initial stimulus (R = 0.971 by linear regression). Such a quantitative relationship would have been more difficult to establish in control PMNs because the level of Mac-1-dependent adhesion to KLH following a single 10 nM fMLF stimulus is already nearly 50%, and even after a 5- to 6-fold up-regulation of Mac-1 surface expression by an initial stimulus, the range available for increased adherence to KLH after a second stimulus would be limited to less than 2-fold. The findings with the patient's cells would appear to show that the amount of Mac-1 on the PMN surface at the time of a given chemoattractant stimulus, whether resident surface Mac-1 or Mac-1 previously translocated to the PMN surface, is directly proportional to the capacity for Mac-1-dependent ligand binding induced by that stimulus.
The patient considered here is unique as the only example known to us of LAD-1 associated with a mutation that specifically involves the CD18 cytoplasmic domain. He also exhibits one clinical feature that is, to our knowledge, unique among patients with LAD-1 in that he tends to form hypertrophic scars after skin injury rather than the atrophic scars observed in other patients with LAD-1.1,7 It remains to be determined if there is a link between these 2 unique features and if the CD18 cytoplasmic domain may play a role in regulating wound healing. Infiltrating leukocytes are an important feature of healing wound tissue, and PMNs, lymphocytes, and macrophages may be sources of transforming growth factor One historic issue deserves comment. The above-cited 1979 report involving this patient8 implicated the overgrowth of Capnocytophaga in the oral cavity in the pathogenesis of PMN chemotactic dysfunction before LAD-1 was a recognized disorder. The documentation of LAD-1 in this patient should encourage a reassessment of this clinical association. Since the PMN granule protease cathepsin G has been found to be a critical constituent in killing of Capnocytophaga,47 it seems likely that impaired delivery of PMNs to gingival tissue would be the cause rather than the consequence of the overgrowth of this organism.
The authors wish to thank Leo Mendoza for assistance with analysis of DNA sequence data, Dr David Kaplan for preparation of lymphocyte lines, and Bonnie J. Hughes for expert technical assistance.
Submitted August 13, 2003; accepted September 22, 2003.
Prepublished online as Blood First Edition Paper, September 25, 2003; DOI 10.1182/blood-2003-08-2780.
Supported by United States Public Health Service (USPHS) grants AI19031, AI45802, and HL42550.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: C. Wayne Smith, CNRC/Leukocyte Biology, 1100 Bates Ave, Suite 6014, Houston, TX 77030; e-mail: cwsmith{at}bcm.tmc.edu.
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Abstract
Introduction
RII, clone IV.3, from Dr Paul Guyre, Hanover, NH), anti-L-selectin (anti-Leu-8; Becton Dickinson), anti-E-selectin from clone CL2,
-E (Invitrogen) were transformed by electroporation using the Gene Pulser II System (Bio-Rad Laboratories, Hercules, CA) under conditions recommended by the vendor for this bacterial strain. The transformed bacteria were added to 1 mL room temperature SOC medium and shaken at 225 rpm for 1 hour at 37°C, serially diluted with SOC medium, and plated on Luria-Bertani (LB) agar plates containing 100 µg/mL ampicillin. 
