Bovine Leukocyte Adhesion Deficiency: In vitro Assessment ofNeutrophil Function and Leukocyte Integrin Expression

Timothy W.J. Olchowy, Philip N. Bochsler, Nancy R. Neilsen, Matthew G. Welborn and David 0. Slauson

ABSTRACT RESUME INTRODUCTION

Bovine leukocyte adhesion defi-ciency (BLAD) was identified in atwo-month-old Holstein heifer calfusing DNA-polymerase chain reac-tion analysis of the affected calf andother clinical parameters. Neutro-phil integrin expression (CD18,CD11a, CD11c), aggregation, andtransendothelial migration werestudied in vitro. Neutrophils wereisolated from the affected calf andfrom normal, healthy, age-matchedcontrol Holstein calves. Neutrophilsisolated from the affected BLADcalf had decreased expression ofleukocyte integrins on their cell sur-face, decreased ability to aggregatein response to chemotactic stimuli,and decreased ability to migrateacross bovine endothelial cell mono-layers in vitro. Transendothelialmigration of neutrophils from nor-mal calves was reduced to levelscomparable to the BLAD neutro-phils by treatment with an anti-CD18 monoclonal antibody (MAb60.3). Peripheral-blood lympho-cytes from the BLAD calf alsoexpressed negligible levels of leuko-cyte integrins, similar to their neu-trophil counterparts. Our experi-mental findings in vitro correlatewell with the clinical observationsof decreased leukocyte traffickingand diminished host defense inleukocyte adhesion-deficient ani-mals. The syndrome of BLAD maybe a suitable model for one of thehuman leukocyte adhesion defi-ciency disorders.

La de'ricience du facteur d'adhe-sion des leucocytes (BLAD) a eteidentifiee chez un veau Holsteinfemelle age de deux mois a l'aide dela technique d'amplification enchaine par la polymerase de l'ADNet differents parametres cliniques.Des tests in vitro sur l'expression del'inte'grine (CD18, CD11a, CD11c),de l'agregation et de la capacite 'atraverser l'endothelium vasculaireont ete realises sur les neutrophilesde l'animal affecte ainsi que chezdes veaux temoins normaux dumeme age. Les neutrophiles del'animal affecte ont demontre unediminution de l'expression de l'int&grine sur leur surface cellulaire,une diminution de leur capacite aagreger suite a un stimuli chemo-tactique ainsi qu'une diminution deleur capacite a migrer a travers unemono couche de cellules endo-theliales. Les neutrophiles temoins,lorsque traites avec un anticorpsmonoclonal anti-CD18, avaient unecapacite migratoire semblable 'acelle des neutrophiles de l'animalaffecte'. L'intetgrite lymphocytaireetait aussi reduite chez l'animalatteint. Les resultats des testsin vitro correlent bien avec l'evalua-tion et l'image clinique de l'animalet ce syndrome rencontre chez lebovin pourrait servir de modelepour l'etude d'un syndrome simi-laire chez l'homme. (Traduit par DrPascal Dubreuil)

Circulating polymorphonuclearleukocytes, including neutrophils,respond quickly to many inflamma-tory stimuli in an attempt to providedefense against invading organisms(1). Neutrophils play a critical role inthe host defense system by phagocy-tosing and destroying invadingmicroorganisms (2,3) using potentmicrobicidal armament such as reac-tive oxygen species, antimicrobialpeptides, and degradative granuleenzymes. Not surprisingly, a defect inneutrophil function would likelyresult in increased susceptibility tobacterial pathogens (3-5).

In response to tissue invaders, theperipheral blood neutrophils execute aprogrammed series of events thatinclude sensing a chemotactic agentor inflammatory stimulus, adhesion tothe vascular endothelium, migrationthrough the vessel wall, chemotaxiswithin the extracellular matrix of tis-sue toward the inflammatory focus,and finally reacting directly to theoffending agent. A critical event inthis process is the adhesion of neu-trophils to the vascular endotheliumvia interaction of the CD 18/CD 11glycoprotein heterodimers on the neu-trophil with the adhesion molecules ofthe endothelium (6-8). DeficientCD18/CDl1 expression on neu-trophils severely limits the adhesiveevent, restricting neutrophil move-ment out of blood vessels andmarkedly reducing the ability of thehost to combat infectious organisms(3,8-10).One of the recently recognized

human leukocyte adhesion disordershas been linked to a genetic defect in

Department of Rural Practice (Olchowy, Welborn) and Department of Pathobiology (Bochsler, Neilsen, Slauson), College of Veterinary Medicine,University of Tennessee, Knoxville, Tennessee 37901-1071.

Supported in part by the University of Tennessee Centers of Excellence, USDA 90-37265-5611, and the Department of Rural Practice, College ofVeterinary Medicine, University of Tennessee.

Submitted April 23, 1993.

Can J Vet Res 1994; 58: 127-133 127

the expression of the CD 18 integrin, acomponent of a neutrophil hetero-dimeric cell-surface receptor (4,5). Thedefect decreases the initial adhesive/rolling event of neutrophil alongendothelial cells, the firm adhesion toendothelium and the subsequentmigration through blood vessel wallsinto the tissue (7,11,12). Humans withthis disorder have severe recurrentinfections characterized by pneumonia,gingivitis, periodontitis and cellulitiswithout pus formation (13). A geneticdeficiency of the CD 18 portion of theneutrophil heterodimer has beenrecently reported in cattle (bovineleukocyte adhesion deficiency orBLAD) (14,15).

In this report we have used in vitroassays to compare the ability ofbovine neutrophils isolated from nor-mal animals and from a naturallyoccurring case of BLAD to migrateacross bovine endothelial cells. Inaddition, we compared neutrophilaggregation responses and leukocyteintegrin expression on neutrophils andlymphocytes of normal and BLADcalves.

MATERIALS AND METHODS

ANIMALS

A four-week-old female Holstein-Friesian calf was identified as a prob-able BLAD animal. Three agematched control female calves wereselected from the same Holstein-Friesian herd. All animals werehoused in individual hutches and fedmilk replacer, calf starter, hay andfree choice fresh water according tothe usual herd management. All pro-tocols were preapproved by theUniversity of Tennessee Animal Careand Concerns Committee.

LABORATORY REAGENTS

Materials were purchased as fol-lows: Zymosan A and hrC5a (SigmaChemical Co., St. Louis, Missouri);fluorescein isothiocyanate-labeledgoat antimouse IgG-IgA-IgM antibody(#55499, Organon Teknika-Cappel,Malvern, Pennsylvania); mouse mye-loma protein IgG2a kappa light chain(#50328, Organon Teknika-Cappel);anti-MHCl mouse monoclonal anti-body (MAb) isotype IgG2, (H58A,VMRD Inc., Pullman, Washington);

anti-CD 1lla mouse MAb of isotypeIgG, (BAQ30A, VMRD); anti-CD1icmouse MAb of isotype IgM(BAQ 1 53A, VMRD); antilymphocytemouse MAb of isotype IgG3 (TH18A,VMRD), anti-CD18 mouse MAb ofisotype IgG2a (MAb 60.3, a kind giftfrom Dr. John Harlan, University ofWashington, Seattle, Washington);Na25'CrO4 in sterile normal saline(5 mCi/mL) (#62015, ICN Pharma-ceuticals, Inc., Irvine, California).Zymosan-activated serum (ZAS) wasprepared from fresh pooled bovineserum using standard methods (16).The ZAS was tested for neutrophilstimulatory activity by aggregometry(17) and stored in 1.0 mL aliquots at-70°C until use.

NEUTROPHIL ISOLATION

Blood (60 mL) was collected byjugular venipuncture using acidcitrate dextrose (1:10) as the antico-agulant. Total and differential leuko-cyte counts were performed on bloodsamples using routine hematologicalmethods. All control calves had nor-mal hematological values. Neutro-phils were isolated by differentialcentrifugation combined with flashhypotonic lysis of the red blood cells(16). The final neutrophil suspensionwas adjusted to a concentration of15 x 106 cells/mL (for antibody label-ing) or 11 x 106 cells mL (aggrega-tion) in HBSS containing Ca2+ andMg2+ (HBSS-CM); or 20 x 106cells/mL in HBSS for 5Cr labelling.The final cell preparations, as verifiedon Wright-Giemsa stained cytocen-trifuge preparations, comprised atleast 95% neutrophils, with theremaining cells being lymphocytes.Cell viability was high (>98%) judgedby trypan blue dye exclusion.

IMMUNOFLUORESCENCE FLOWCYTOMETRY

The expression of bovine neutro-phil integrins was assessed usingmonoclonal antibodies that recognizethe common P2 integrin subunit CD 18(MAb 60.3) and the leukocyte inte-grin a-subunits, CD 1 IA and CD11 c.MAb 60.3 is a mouse-antihumanIgG2a that recognizes the CD 18 multi-meric cell surface glycoprotein com-plex (18). The specificity of MAb60.3 for bovine neutrophils has beenverified (19).

Isolated bovine neutrophils werekept at 4°C in HBSS-CM. All incuba-tion steps were at 37°C. To minimizenonspecific Fc binding, neutrophilaliquots of 400 pL (15 x 106 neutro-phils/mL) were suspended with 50 pLof mouse myeloma IgG2. kappa lightchain (20 ,ug/mL) in siliconized500 pL Eppendorf tubes and incubatedwith occasional mixing for 10 min.Fifty microliters (200 p,g/mL) ofmonoclonal antibody (anti-CD18, anti-CD 11 a or anti-CD 1 I c) was added andthe tubes reincubated. Cells were fixedfor S min with 25 pLL of 10% para-formaldehyde and centrifuged (200 x g,2 min). Supernatants were discardedand the pellets washed twice with400 pL of 1% heat inactivated bovineserum albumin (Sigma). Pellets wereresuspended in 400 ,L of 10% normalgoat serum (Sigma), incubated for10 min, centrifuged and resuspended in400 pL of FITC labeled goat-antimouse IgG-IgA-IgM (FITC-GAM).The cell preparation was incubated for30 min, centrifuged and resuspended inHBSS-CM in preparation for analysis.Immunofluorescence flow cytometrywas performed using a FACScan flowcytometer (FACScan, Becton Dicken-son, Braintree, Massachusetts). Datawas acquired by counting 10,000 cells/events for each treatment per experi-ment, and was expressed as relativefluorescence intensity units aftersubtracting nonspecific (control)fluorescence.Buffy coats collected from cen-

trifuged blood samples, were used asa source of mixed leukocyte prepara-tions containing lymphocytes. Thelymphocyte population was identifiedby its low forward and side scatterand by positive indirect immunofluo-rescence labeling of cells in a paralleltreatment using a monoclonal anti-body (TH1 8A) recognizing bovine Tand B lymphocytes and null cells.These lymphocytes were treated withMAbs similar to the neutrophils toassess leukocyte integrin expression.The BLAD values were compared tolymphocytes from normal controlcalves.

In some experiments, the potentialupregulation of CD18 expression bythe BLAD calf neutrophils wasassessed with an additional 10 minincubation in the presence of 10%ZAS prior to antibody labeling.

128

TABLE I. Detection of adhesion related surface antigens CD11a, CD11c and CD18 on bovineneutrophils and lymphocytes from normal control calves (NORMAL) and the BLAD calf(BLAD). Relative values represent the monoclonal-antibody associated fluorescence intensityof the neutrophils or lymphocytes. The nonspecific fluorescence has been subtracted. "BLA"indicates bovine lymphocyte antigen. "-" indicates assay not performed

NeutrophilsNORMAL Lymphocytes

Antigen BLAD Calf I Calf 2 Calf 3 BLAD NORMAL

CDI laCDI lcCD18BLA

f

2.310.947.3

1380.4182.7

1848.3 1050.5 949.5

73.441.732.3

3698.6

Relative Fluorescence Intensity (log)Fig. 1. Representative indirect immunofluorescence flow cytometric analysis of anMAb binding to normal control or BLAD neutrophils. "f" indicates the relative nuevents counted by the flow cytometer. Relative fluorescence is in log scale.

2545.5125.9

2297.73739.1

plates were incubated in a humidifiedchamber (90 min, 37°C). The trans-wells were then removed. Neutrophilsthat had migrated across the endothe-lium into the tissue plate well andneutrophils that remained in the trans-well were lysed with Triton X-100(0.1%, 30 min). The 5Cr-neutrophil-associated disintegrations per minute(cpm) were determined by gammacounting (Cobra 5005, Packard Instru-ment Co., Meriden, Connecticut). Thecpm ratios were used to calculate theneutrophil migration which wasexpressed as the percent of total neutro-phils loaded into the transwell.

NEUTROPHIL AGGREGATION

Neutrophil aggregation was semi-quantified using a standard plateletaggregometer and strip recorder asdescribed (17). Immediately followingthe aggregation experiments, neutro-phils were fixed with 0.5% para-formaldehyde and sample aliquots werevisually assessed for the presence/absence of aggregation using lightmicroscopy, and then photographedusing standard light microscopy.

DNA-POLYMERASE CHAIN REACTION(PCR) ANALYSIS

A whole blood sample from the clin-ically affected calf was collected into

T an EDTA vacutainer tube and shipped104 to Dr. M.E. Kehrli Jr, at the National

Animal Disease Center (USDA-Agricultural Research Services, Ames,

ti.CD18 Iowa) for DNA-PCR analysis (14,15).Imber of

TRANSENDOTHELIAL NEUTROPHILMIGRATION

Isolated neutrophils suspended inHBSS were labeled with 5Cr (asNa,CrO4; 3 ,uCi/106 neutrophils) for60 min and washed (20). Culturedbovine aortic endothelial cells weregrown to confluency on 6.5 mmtissue-culture-treated polycarbonatemembrane transwell inserts (Trans-well, #3415, Costar, Cambridge,Massachusetts) perforated with3.0 ,urm diameter pores, and nestedwithin 24-well tissue culture plates.Confluency of endothelial cells wasassessed by inhibited movement ofFITC-labeled immunoglobulins acrossthe endothelial cell layer.

The chemotaxins hrC5a (l0-7Mfinal concentration) and ZAS (10%final concentration) were suspendedin DMEM (Dulbecco's ModifiedEagle's Media with 4.5 g/L glucose,without L-glutamine, WhittakerBioproducts, Walkersville, Maryland)and 100 puL added to the appropriatetissue plate well. 5'Cr-labeled neu-trophils (5 x 105) in 100 puL wereadded to the transwells above theendothelial cell monolayers and coin-cubated with 100 p,L of DMEM or100 ,u.L (20 jiwg/mL final concentra-tion) of anti-CD18 MAb (MAb 60.3),mouse myeloma MAb (antibodyspecificity control), or antibovineMHC-l MAb (H58A) (control). The

RESULTS

CLINICAL FINDINGS

The BLAD calf had clinical signsof disease including small stature, lowbody weight, poorly formed soft fecesto diarrhea, gingivitis and periodonti-tis which was most severe around theincisors. Repeated complete bloodcounts of the BLAD calf demon-strated a marked neutrophilic leuko-cytosis with a total white blood cellcount of > 60,000/,uL and a neutrophilcount of > 40,000/jiL.

DNA-PCR ANALYSIS

The DNA-PCR analysis indicatedthat the clinically affected calf was

homozygous for the BLAD genetic

129

CHEMOTAXINS

80

70

60

50

40

30

20

10

0None None M.M. CD18 MHC1 None M.M. CD18 MHC1

Antibody Treatment of NeutrophilsFig. 2. In vitro transendothelial neutrophil migration. Values are means of two experimentsand represent the percentage of neutrophils which migrated across the endothelial mem-brane. Solid bars represent neutrophils from normal calves (n = 2). Hatched bars representBLAD calf neutrophils. hrC5 = human recombinant C5a. ZAS = 10% zymosan activatedserum. M.M. = mouse myeloma MAb (antibody specificity control). CD18 = anti-CD18 MAb(MAb 60.3). MHC1 = MAb to the major histocompatibility complex 1 (control). Error barsequal one standard deviation of the mean. The apparent effect of MAb 60.3 on the normalcalves' neutrophils was diminished in the presence of hrC5a due to one outlying data point.

Bind CaltftT

0

Stimulus60

ndIS

tim -

Fig. 3. Aggregometer tracing from ZAS-stimulated normal and BLAD neutrophils. Tracingrepresents typical aggregation response of BLAD and normal control calf neutrophils to stim-ulation. Arrow indicates time point when ZAS was added to cell suspension. T = light trans-mittance, proportional to aggregation.

defect identified as the D128G allelemutation.

IMMUNOFLUORESCENCE FLOWCYTOMETRY

Neutrophils from the BLAD calfhad a marked reduction in CD18,CD I c and CD I a leukocyte integrinexpression compared to normal calfneutrophils (Table I). Lymphocyteexpression of CD18 was similarlyreduced in the BLAD calf comparedto the normal calves (Table I). Someincreased fluorescence appeared on asmall population of neutrophils fromthe BLAD calf and was attributed tononspecific binding of labeled anti-body to Fc receptors on a subpopula-tion of neutrophils (Fig. 1). The posi-tioning (forward scatter, side scatter)indicated that this discrepancy wasnot caused by the formation of dou-blet or triplet cell clusters.

In preliminary experiments, CD18expression by neutrophils from theBLAD and control calves (n = 1) wasnot remarkably increased following10% ZAS stimulation.

TRANSENDOTHELIAL MIGRATION

Fewer neutrophils collected fromthe BLAD calf underwent transendo-thelial migration compared to the nor-mal calf neutrophils (Fig. 2), but someneutrophil migration occurred in allwells. There was a difference in theextent of the neutrophil migratoryresponse to the chemotaxins withZAS inducing a higher percentage ofneutrophil migration in all calves.The percentage of control calves'

neutrophils migrating across theendothelial monolayer in response tothe chemotaxin ZAS was reduced tothe level of the BLAD calf neutro-phils after MAb 60.3 treatment. Thereduction in the migration of the con-trol calves' neutrophils toward thehrC5a chemotaxin was not as pro-found. The presence or absence of anyantibody had no effect on the rela-tively poor migratory response of theneutrophils from the BLAD calf.

NEUTROPHIL AGGREGATION

The BLAD neutrophils stimulatedwith 10% ZAS demonstrated markedlyreduced aggregation activity comparedto normal bovine neutrophils (Fig. 3).This was also evident morphologicallyas the neutrophils from the normal

C00

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:3

z

130

control calves showed clear evidenceof aggregation, whereas neutrophilsfrom the BLAD calf displayed mini-mal aggregation (Fig. 4).

DISCUSSION

The syndrome of bovine leukocyteadhesion deficiency was initially sus-pected in this calf because of themarked peripheral blood neutrophilia.The very low level of integrin (CD18)expression on neutrophil and lympho-cyte plasma membranes detected byFACS analysis and the results of theDNA-PCR analysis confirmed thediagnosis (14,15). The DNA-PCRanalysis indicated that this calf washomozygous recessive for the geneticdefect in CD18 production (14,15).Investigation of the calf's pedigreeconfirmed the presence of BLAD car-rier animals in the dam's backgroundwith the immediate sire being aknown carrier of the BLAD trait. Eli-mination of this genetic defect fromthe Holstein-Friesian breed is alreadyin progress using the DNA-PCR assayand the extensive records of the breedassociation.Our FACS data demonstrated a

marked reduction in the expression ofthe CD 18, CD I I a and CD 1 c sub-units on the surface of the BLADneutrophils. The interpretation of thedata required an assumption that theCD18 integrin was constitutivelyexpressed on the cell surface. Con-stitutive expression has previouslybeen demonstrated in human andbovine neutrophils (13,19). The stim-ulated (ZAS, C5a) human neutrophilswere capable of integrin upregulationon plasma membrane (4), but theresponse of bovine neutrophils wascomparatively much smaller (19). Theupregulation on human neutrophilswas believed to be the result of trans-location of intracellular integrin stor-age pools to the cell surface (4).Our preliminary data indicated very

little quantative upregulation of CD18occurred on bovine neutrophils inresponse to ZAS and was consistentwith previous findings (19). It may bethat bovine neutrophils do not quanti-tatively upregulate the leukocyte inte-grins to a significant degree, butinstead rely on qualitative, conforma-tional changes in the integrins for

*88veews S; - M

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:

iSs

:l..>R

,4

M

si

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.:

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Fig. 4. Photomicrograph of neutrophils from typical aggregation experiment. Normal controlcalf neutrophils before (A) and after (B) addition of ZAS. BLAD calf neutrophils before (C)and after (D) addition of ZAS.

enhanced adhesiveness. Furtherstudies are required to explore thispossibility.

There was a reduced expression ofleukocyte adhesion integrins on thelymphocytes from the BLAD calf. Toour knowledge this is the first reportof reduced integrin expression (pre-sumably a CD 1 I a/CD 18 or CD 1 I a/CD 18-like molecule) on bovine lym-phocytes. Low lymphocyte integrin(CD1 la/CD18, CDI lb/CD18, CD1 Ic/CD18) expression has been docu-mented to occur in human LADpatients (4). Our finding may be sig-nificant when considering the mecha-nism(s) of reduced lymphocyte func-tion in human LAD patients.

In regard to the function of BLADcalf lymphocytes, only a single reportof unaffected mitogen stimulatedblastogenesis presently exists (14).Reduced lymphocyte blastogenesisreponses to phytohemagglutinin, con-canavalin A and pokeweed mitogenhave been reported in a dog with adeficiency of the neutrophil surfaceglycoproteins CD11b, CD11a andCD18 (21). Only the dog's neutro-phils were examined for surface inte-grin expression. Humans with LADhave deficient T-lymphocyte medi-

ated killing, lymphocyte proliferativeresponses, natural killing activity andantibody-dependent killing (22-24).The migration of neutrophils across

endothelial cells is dependent upon thepresence and interaction of cell-sur-face receptors (10). In our in vitromodel we demonstrated a reduction inneutrophil transendothelial migration,indicating the importance of CD18integrin expression. This is consistentwith the reduced 24 h neutrophil accu-mulation in subcutaneously implantedsponges in rabbits pretreated with aMAb to CD18 (9). In vitro studieswith neutrophils from BLAD calveshave documented a reduction in manyof the cellular functions (14,25,26)but have not addressed the essentialfunction of transendothelial migrationrequired before the neutrophil cancomplete its antimicrobial function inbody tissues.The transendothelial migration

model described herein has been usedpreviously to investigate the migra-tion response of normal neonatal andadult bovine neutrophils to chemotac-tic stimuli (19). Our model suffers,like most in vitro systems, from theinherent limitation of excessive sim-plicity relative to the in vivo environ-

131

ment. However, the model does createa neutrophil-endothelial interactionsimilar in many respects to the in vivoenvironment and thereby facilitatesthe use of monoclonal antibodies inthe study of some cellular interac-tions. Monoclonal antibodies specificfor the leukocyte integrin CD18 onnormal neonatal and adult bovine neu-trophils have a marked adverse impactupon transendothelial migration (20).The migration of normal calf neu-

trophils was reduced by the mono-clonal antibody directed againstCD18, confirming the critical impor-tance of this molecule in the neu-trophil-endothelial interaction (20).Failure of the monoclonal antibodyspecific for bovine MHC-1 and thenonspecific mouse myeloma antibodyto alter neutrophil transendothelialmigration indicated that inhibition ofmigration induced by MAb 60.3 wascaused by very specific blocking ofthe endothelial cell-CD18/CD11interaction. The neutrophils of theBLAD calf were unaffected by thetype of antibody treatment exhibitingnone of the migration inhibitionobserved with the normal calf neutro-phils. Since the BLAD calf did notexpress CDI8/CD1 1 on its neutro-phils, the failure of monoclonal anti-body directed against CD18 to inhibitneutrophil transendothelial migrationwas not surprising. The results of thisstudy are very similar to studies inhumans (8,10) demonstrating theimportance of the CD 18/CD 11 hetero-dimer in transendothelial migration ofneutrophils. The data herein also sup-port the proposal of Kehrli that cattleafflicted with bovine leukocyte adhe-sion deficiency be used as a model forstudy of the human leukocyte adhe-sion deficiency genetic disorder (27).There was a difference in the

response of the neonatal bovine neu-trophil to hrC5a and ZAS in ourmodel. Chemotaxins which stimulatedirected migration include comple-ment components (C5a, C3b), bacterialproducts (Escherichia coli bacterialfiltrate) and zymosan activated serum(28). Since hrC5a is a purified productand ZAS is a product containing amultitude of known and unknown fac-tors (including C5a), it is not unex-pected that ZAS might stimulateincreased neutrophil chemotaxis, as it

132

did in our experiments. A portion ofthe variation may be due to differencesin biological activity between hrC5aand naturally produced bovine C5a andC5ades Arg' both of which would bepresent in ZAS.

Neutrophil aggregation is a stimulus-associated, integrin dependent responseof the neutrophil (4), and the reducedaggregation of BLAD neutrophils isconsistent with previous work in LADhumans (29). Reports in horses and cat-tle have demonstrated reduced neu-trophil aggregation after blocking cell-to-cell CD18-like interaction withspecific monoclonal antibody (19). Inaddition, the PMA-induced aggregationresponse of neutrophils obtained froman integrin deficient dog was markedlydepressed compared to normal controldogs (21). Adhesion dependent pro-cesses, which include not only aggrega-tion but also spreading, orientation inchemotactic gradients, ADCC andphagocytosis of particles (29), wouldalso be expected to be decreased inBLAD calves and human LADpatients. Adhesion independent func-tions such as shape change, superox-ide generation, secretion in responseto soluble stimuli, membrane fluidity,surface charge and microtubuleassembly (29) should be minimallyaltered by deficient CD18/CD 1Iexpression.

In vitro studies with BLAD neu-trophils have demonstrated reductionsin cell functions (14,25,26,30). Theneutrophil functions most severelyaffected are those requiring or associ-ated with cell adhesion (31). There-fore the type of cell function assay(adhered versus suspended cells) mayhave a marked impact on experimen-tal results. Opsonized particles arepoorly phagocytosed by CD18 defi-cient neutrophils and therefore wouldbe a poor assessment of respiratoryburst activity (29,32). An apparentfunctional defect in CD18 deficientneutrophils may be observed if thein vitro assay system relied uponadherent neutrophils. Our model mim-ics the in vivo system requiring theadherence of neutrophils to endothe-lial cells.

In human LAD patients, all leuko-cyte cell types (lymphocytes, mono-cytes, granulocytes) have reduced sur-face expression of the leukocyte

adhesion integrin glycoproteins (4,5).In BLAD calves only the neutrophilhas previously been identified as defi-cient in integrin expression (14). Ourfinding of deficient leukocyte adhe-sion integrin expression on peripheralblood lymphocytes confirms the logi-cal expectation from the earlier mole-cular definition of the BLAD syn-drome (14).We have found that there is a

marked impairment of neutrophiltransendothelial migration in BLADcalves, which has significant correla-tion with clinical findings such asimpaired defense against microorgan-isms. Because this condition has beengenetically defined in cattle andappears to be similar to the humandefect, and because reproductivemanipulation of cattle is routine andethically acceptable, the BLAD calfappears to be a likely candidate inmodeling human leukocyte adhesiondeficiency.

ACKNOWLEDGMENTS

Grateful acknowledgment is givento Dr. John Harlan, University ofWashington, Seattle for his generousgift of monoclonal antibody 60.3.

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