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Kindlin-3 in hematopoietic integrin activation: Absence in leukocyte adhesion deficiency type III van de Vijver, E.

Publication date 2013

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Citation for published version (APA): van de Vijver, E. (2013). Kindlin-3 in hematopoietic integrin activation: Absence in leukocyte adhesion deficiency type III.

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Download date:02 Oct 2021 CHAPTER 3

Leukocyte Adhesion Deficiencies

Edith van de Vijver, Timo K. van den Berg, Taco W. Kuijpers

Hematol Oncol Clin N Am. 2013 Feb; 27(1): 101-116 Abstract During inflammation, leukocytes play a key role in maintaining tissue homeostasis by elimination of pathogens and removal of damaged tissue. Leukocytes migrate to the site of inflammation by crawling over and through the blood vessel wall, into the tissue. Leukocyte adhesion deficiencies (i.e., LAD-I, -II, and LAD-I/variant, the latter also known as LAD-III) are caused by defects in the adhesion of leukocytes to the blood vessel wall, due to mutations in the genes encoding β2 integrin (ITGB2), a GDP-fucose transport protein (SLC35C1) and kindlin-3 (FERMT3), respectively. Patients experience recurrent nonpussing bacterial and , often preceded by delayed separation of the umbilical cord, and additional symptoms depending on the subtype. For LAD-I and LAD-III, the only curative treatment is hematopoietic stem cell transplantation. In case of LAD-II, oral fucose supplementation may invert the immune defect, but additional mental retardation is hardly improved. 3 Leukocyte Adhesion Deficiencies 35

4;5 Extravasation is a multi-step 1;3 These gradients of chemotaxins 2 Leukocytes migrate to the site of inflammation Chemotaxins may be derived either from the 1 Integrins are type I transmembrane glycoproteins that that glycoproteins transmembrane I type are Integrins 1;6 eukocyte recruitment and extravasation. L Within thelocal environment inflammatoryanof tissuereaction, the endothelium begins 4 Leukocytes following the chemotaxin gradient towards the site of have to leave In contrast to the low-avidity binding of leukocytes to selectins, the final step of firm adhesion firm of step final the selectins, to leukocytes of binding low-avidity the to contrast In igure 1. ntroduction the blood stream, in a process called extravasation (Figure 1). recruit the leukocytes in interplay with factors expressed locally on the luminal vessel side endothelialof bloodcells. are short-living leukocytes that are recruited early in inflammatorythe response. and and subsequent migration depends on stable interaction between integrins on the leukocytes cells. endothelial the on ligands their and F following a gradient of chemotaxins. The cells slow down due to transient interactions between selectins andtheir glycosylated ligands, which aredefective in LAD-II. Next, stable adhesion by leukocyte integrins, absentLAD-I,inligands endotheliumthe toon resultsleukocytein arrest. Activation bloodofcellintegrins is decreased in LAD-III. Healthy neutrophils extravasate subsequent to firm adhesion. I Leukocyte recruitment and extravasation Duringinflammation, circulating leukocytes infection of site the migrateto following gradient a of chemotaxins in a process called . process involving adhesion molecules, in which chemotaxins function as activating agents (pro-)inflammatoryor extravasationmediators. of step first The consistsinitial contactof between endothelial cells and leukocytes marginated by the fluid flow of the blood. L-selectin (CD62L) on leukocytes plays a role herein, contacting several cell adhesion cells.molecules on endothelial infected tissue or local complement activation, or directlyand from diffusethe pathogens withinthemselves, the tissue into the local vasculature. to expresstoadhesionthe molecules P-selectin (CD62P) E-selectinandlateron (CD62E). Thelow- avidity interaction of these selectins with their fucosylated ligands on the opposite cells forces the leukocytes to slow down and start a rolling movement along the vessel wall (Figure 1). 2 β D α subunit: The head α 13;14 Since adhesion 9 The head domain 4 2 integrins, resulting β 8

2 integrins bind to adhesion adhesion to bind integrins 2 12 subunit. β β subunits, with sizes of 120-170 kDa kDa 120-170 of sizes with subunits, and β α 2 (gp150,95; CD11c/CD18); and CD11c/CD18); 2 (gp150,95; β as a result of chemokine binding to X and and subunits form 24 known combinations, α α β e.g. and 8 α Moreover, for continued sensing of the chemotaxin 10;11 2 (CR3; CD11b/CD18), 2 (CR3; CD11b/CD18), β M Leukocyte activation, α In mammals, 18 7 7;12 2 integrins2 areessential for the connections with the tissue cells orwith β 2 2 integrin receptor subfamily is selectively expressed on leukocytes and β 2 integrins, leading to an enhanced affinity for their ligands (“inside-out” signaling). 2 (LFA-1; CD11a/CD18), CD11a/CD18), 2 (LFA-1; β β L As mentioned above, slowly rolling leukocytes are able to recognize concentration The common activator of most, if not all, integrins is talin, a large cytoskeletal protein that α moleculessuch asthe theextracellular matrix proteins, these connections must be formed at the front of the moving leukocytes and broken at the rear end. chemokinereceptors, ligandbinding selectins, to antigenorand binding receptor, theT-cell to subsequent intracellular signaling induces conformational changes in the extracellular regions of the In addition, integrins cluster in larger complexes, which increases their ligand avidity. Binding to extracellular ligands leads to further conformational changes of the in high ligand affinity and subsequent recruitment of cytosolicdownstreamproteins andsignaling the initiationcascades of that regulate cell spreading proliferation,and differentiation and alterapoptosis (“outside-in” genesignaling). expression, cell domainoftalin protein,contains ezrin, FERMaradixin, (4.1 moesin) domain, consisting ofthree subdomains, F1, F2 and F3. The latter, the F3 subdomain, contains a phosphotyrosine-binding(PTB)-like domain motifthatbinds foundthe NPxY/F to the inmembrane-proximal cytoplasmic is attached to the membrane via two flexible legs(one intracellularlyfrom each subunit), as whichterminate short cytoplasmic their tails. ability This to domain transduce architecture bidirectional and of “outside-in” signals (Figure integrins across2). underlies the plasma membrane: “inside-out” gradient, the chemotaxins must dissociate from their respective receptors for repeated usage. This occurs through internalization of the ligand-receptor of complex, the intracellularconnection, disruptionand transport of the reappearancefree receptorof theto free the receptor front on of the the leukocyte cell, surface.chemotaxin followed gradientWithin persists and leukocyteby the migration isinfected maintained. tissue, the and and 90-130 kDa, respectively. form heterodimers via non-covalent association of their their of association non-covalent via heterodimers form Integrin activation Theligand specificity of integrins is determined by their large extracellular ligand-binding head domain, which is composed of several domains of both the differences in a gradient of chemotaxins andto direct their movementtowards the sourcethese of agents. Although the details of this process remain unknown, causesthe a differencegradient in the numbermost of ligand-boundlikely chemotaxinreceptors on either side of the cell,thereby inducing the cytoskeletal rearrangements needed for movement. acts as an allosteric activator of integrins by inducing their ligand-binding affinity. each of which can bind to a specific repertoire of cell-surface, protein extracellular ligands. matrix The or soluble comprises four differentheterodimeric proteins, eachof which contains a different i.e. molecules on endothelial cells (intercellular adhesion molecule [ICAM]-1 and ICAM-2), and to CR3. is neutrophils on integrin b2 main The factors. complement several (CD11d/CD18), the latter only being expressed on . The The macrophages. on expressed being only latter the (CD11d/CD18), 36

3 Leukocyte Adhesion Deficiencies 3 Leukocyte Adhesion Deficiencies 37

20;21 , activated by In a study on α 12;16 chain, consisting of an β and a α 3, it was proposed that PKC β In man, the family consists of three members 18;19 Since kindlins possess a FERM domain that is 18

12 IIb and α Integrins Integrins are of heterodimers an 3 integrins. β The head domain is connected to a long cytoplasmic rod which 15 17 2 and β 1, β integrins. β by chemotaxin binding, causes “inside-out” signaling. This signaling intracellular – in which e.g. eukocyte eukocyte integrin activation. L While binding of talin appears to be the final step in integrin activation, the activity of talin More recently, kindlin proteins have arisen as key players in integrin activation. Kindlins igure igure 2. – kindlin-1, -2 and -3 – that share a hematopoietic highcell types, degreewhere it playsof an importanthomology. role in Kindlin-3a variety ofis functions expresseddepending integrin-mediatedon inadhesion, suchplateletas clotformation all andleukocyte extravasation. CHO cells expressing platelet integrins itself may be regulated by a variety of cell-type specific signaling pathways. region of several F can interact with the cytoskeleton, allowing talin to contribute substantially motilityto viaadhesion “outside-in” signalingand cascades. compriseafamily of integrin-binding proteins. Biochemical studies have confirmed thatcytoplasmic all tails kindlins of directly bind synthetically generated homologous to that of talin, it was hypothesized that kindlins, like talin, interact with the phorbol-12-myristate-acetatecellreceptor(PMA)Tstimulation, or phosphorylates PKD1,which associates with a small GTPase from the Rap or Ras uponformation activationanof family complex witheffectorits protein Rap1-GTP-interactingthat adaptor in turn activates the integrin molecule (RIAM) and talin. extracellular extracellular ligand-binding head, a transmembrane domain and two activation, short affinity. cytoplasmic ligand increased tails. in resulting Leukocyte change conformational a induces - role important an play kindlin and talin participate may kindlin and talin where signaling, “outside-in” downstream initiates binding ligand Subsequent again, which leads to reversible shuttling to a high Together with affinityconformation. integrin clustering, chemotaxis. and spreading cell as such processes mediated adhesion allows conformation affinity high this

27 25 Fungal 28;29 2 integrins. Biochemical β 22 12 Due to the lack of adhesive 2 integrins on the leukocyte 29 β 2 integrins is found. Decreased β integrins.studiesrecentHowever, β subunit of the leukocyte integrins is distinct from the talin-binding β 2, CD18),2,locatedgenethe21q22.3 at (OMIM β I β As a result, patients with any LAD subtype suffer (integrin 23;24 ITGB2 Infections are usually apparent from birth onward, together eficiency type 25;28 2 integrin. So far, 86 different mutations have beenreported. D 2 subunit leads to a similar decrease in the expression of all four a β β Kindlins are essential for “inside-out” integrin activation and recent 22 3. β dhesion A Mutationsfound inare 26 ocyte k eu with severe septicaemia in some patients, and a common presenting feature is omphalitis with delayed separation of the umbilical cord (Figure 3). Later on patientsnecrotizing developinfections non-purulent,of the skin and mucous membranes, resulting atin early aage. Absencehigh of pus mortalityformation at therate sites of infection is a hallmark and the infections have a high tendency for recurrence; secondary bacteremias may also occur. Among patients who survive infancy, severe gingivitis and chronic peridontitis are major features. studies with mutants of kindlin-2 have shown that the PTB domain in F3 is, in analogyessential to talin,for integrin binding, in addition to a requirement of for the interaction N-terminuswith of the protein from severe bacterial infections and neutrophilia, often the preceded umbilical by cord delayed (Table separation1). ofLAD-II is characterized whereas in LAD-III,by the immune defectsadditional are supplemented withdevelopmental a Glanzmann Thrombasthenia- problems, like bleeding tendency. infections may present in individual cases. LAD-Ipatientsmoderateexhibit ,tomild especially granulocytosis, neutrophilwith counts reaching levels above during 100,000/ml acute infection. Imaging and Additional Testing Physical Examination recurrent,life-threateningLAD-Imanifestsinfections,fungalby primarilybacterial localizedand to skin and mucosal surfaces. L Leukocyte adhesion deficiency type I (LAD-I) isdecreased an autosomalexpression recessive or functioning disorder of causedCD18, by the LAD Leukocyte adhesion deficiencies (i.e., LAD-I,variant) -IIare and caused –III, by defects the in latter the adhesion alsoneutrophils) of leukocytes known to (especially the as blood LAD-1/ vessel wall. membrane-proximal NPxY/F motif in the cytoplasmicthemembrane-proximalof tailinmotif NPxY/F havedemonstrated that the kindlin-binding site of site, i.e. at a membrane-distal NxxY/F motif in the cytoplasmic integrin tail. LAD-Iwasfirst described the in early 80s and since then several hundreds patientsof have been reported. Usually, this leads to the absence or decreased expression of the surface, but sometimes a normal expression of nonfunctional expression of the common subunits on the leukocyte surface. reports point to an additional role in downstream “outside-in” cascades. *600065) that encodes the 38

3 Leukocyte Adhesion Deficiencies 3 Leukocyte Adhesion Deficiencies 39 . ITGB2 up to 6 /ml, of/ml, which III 6 - Low (20%) often prior siblings diseased ++/- (varies per patient) + (potential benefit established) - + + (also prophylactic) + (aggregation) + (firm adhesion, adhesion, (firm + chemotaxis, spreading) + + Plt 20-320 avg140 + + (FERMT3) Hb 6-9/12-14 - + (20.10 70.10 30-90% neutrophils) - LAD + (some omphalitis) + (all have bacterial, some have fungal) /ml during/ml 6 II - High - + ? + - + (rolling) - (SLeX, LeX, H-antigen, N-glycans) + - - + (SLC35C1) - + (up to 150.10 + (mental retardation, autism, convulsions, cerebral atrophy) infections) LAD + (growth retardation, short stature, coarse face) - + (decreasing during childhood; periodontitis) up to 6 /ml) 6 I Gram-negative enteric organisms or fungi. - Severe form: low; moderate form: better - + (potential benefit established) - + (severe vs moderate) + - + (firm adhesion, chemotaxis) + 1-30%, or1-30%, present but dysfunctional + (20.10 - + (ITGB2) - - 140.10 LAD - - + (common omphalitis) ++ (skin & mucosal, gingivitis, periodontitis); necrotizing) esting T

HSCT dditional ptions A O Staphylococcus aureus, Characteristics of LAD-I, -II and -III Definitive diagnosis of LAD-I is based on genetic analysis,revealing mutations in herapeutic hysical examination urvival without able 1. maging and Abbreviations: Avg, average; Hb, hemoglobin; HSCT, hematopoieticsLeX, sialyl Lewisstem X; LeX, Lewiscell X. transplantation; Plt, platelets; S Thrombocyte transfusion transfusion Fucose supplementation HSCT T Antibiotics Platelet defects defects Expression of fucosylated glycoconjugates Expression of integrins (CD18, CD11b) I Leukocytosis Thrombocytosis Genetic defect Bleeding tendency Erythrocytosis Neural abnormalities Dysmorphism Delayed separation of umbilical cord, wound healing defect P Recurrent non-purulent bacterial and fungal infections T capacity only few, if any, leukocytes are present at the sites of infection, which are most often caused by Flow cytometry with antibodies to detect CD18 allows discrimination of two forms of LAD-I, A 30

29;32

31 LAD-I and -III patients commonly present Rebuck skin-window test. in vivo studies on neutrophil functions have demonstrated a marked defect in vitro issue necrosis after removal of the umbilical stump. T Extensive Both reduced-intensity and myeloablative conditioning regimens are currently being used igure 3. in random migration as well as chemotaxistransmigration across toendothelial cell various layers were chemoattractants.found to be severely Adhesionimpaired. Neutrophils to fail to mobilize to skin and sites in the rare third group with severe symptoms exhibits normal expression of a functionally-defective mutant protein. The severity of clinical with presentationthe percentage and of leukocytes complications demonstrating normal in CR3 LAD-Icell surface correlatedegree expression of moleculeand/or deficiency. Patients with severe LAD-I exhibit earlier, more frequent,more and serious episodes of infection, often leading to death a in moderateinfancy, towhereas mild patients phenotype with experience fewer serious infectious surviveepisodes into adulthood. and commonly Therapeutic Options and infections, recurrent or acute treat specifically and prevent to used commonly are Antibiotics patients affectedwith the moderate form may survive to adulthood solely with antibiotics. As a curative treatment, hematopoietic stem cell transplantation (HSCT) is the only approach, and is of form LAD-I. severe the from suffering patients for choice of treatment the often most i.e. a severe form with less than 2% CD18 expression and a moderate form with 2-30%. F in HSCT of LAD-I patients. With myeloablative conditioning, more complete depletion of host marrow can be achieved, thereby decreasing the possibility of mixed chimerism and the risk of rejection.However, pre-transplant infections in immunodeficient patients leadto a high rise in mortality rate with this regimen, especially in patients suffering from comorbid complications. withdelayed separation theumbilicalof cord,oftenfollowed omphalitis,by andresulting tissuein necrosis in some cases. 40

3 Leukocyte Adhesion Deficiencies 3 Leukocyte Adhesion Deficiencies 41

36;37 (Solute carrier family Overall,granulocyte ofrolethe SLC35C1 33 34;35 Survival of HSCT treatment is lower 29;32 II Recipients of RIC transplant, those with either full or mixed 32 eficiency type This protein is encoded by D

40;41 29 As a result,initialtheaAs“rolling”leukocytes ofendothelial the over vessel

38;39 33 38;39 dhesion A ocyte k Further, granulocyte transfusions have been reported as a successful supplementation to The molecular defect in LAD-II has been identified as a deficiency in a Golgi GDP-fucose More recently, gene therapy has arisen as a potential treatment. Although so far gene eu wall in areas of inflammation, which is mediatedon the leukocytes by and reversible E- or P-selectins contact on betweenthe endothelial L-selectins cells with theirfucosyl ligandsrespective on the oppositesialated cells, is disturbed. Without rolling, the leukocytes cannot slow down and stably adhere, and in this way LAD-IIand recruitment to leadsthe site of infection.to Fucosylationdecreased is important leukocyteas well for severalextravasation unrelated functions, and LAD-II patients present as a result with additional symptoms, including mentaland growth retardation. LAD-I treatment. A patient who was suffering for 18 months(EG)lesion,despite treatmentfrom targetedwith antibiotics anti-fungal andanbeencured has therapy, ecthyma gangrenosum massiveby granulocyte transfusions associationin withG-CSF. chimerism, had a long-term survival rate with no manifestation of LAD-I symptoms. transport protein (GFTP). According to studies by the group of Hamidieh et al., use of conditioningthe less (RIC)toxic reduced-intensityregimen is found to be a more safe the treatmentand of feasibleLAD-I patients.therapeutic approach in The rare LAD-II syndrome was firstreported in1992 up into date twoless unrelatedthan 10Arab patients Israeli have boys,been and reported, most of them from the Middle East. L Complications and Concerns Frequent use of antibiotics may result in resistance of the bacteria. HSCT can be unsuccessful especially in case of an incompletely matched donor. Clinical Outcomes The moderate form of LAD-I can often be controlled acute with infectiousprompt use episodesof antibiotics and, during sometimes, from severe prophylacticLAD-I, transplantation antibiotics.is commonly required at For an early age. patientsA multicenter study sufferinginvolving 36 patients demonstrated an overall survival rate of 75%, with best results from HLA- matched stem cell donors. than average for immunocompromised patients owing to the risk of pre-transplant infections. Althoughgranulocyte transfusions haveaddedtreatmentto somecases,in side theeffects may induce additional risks. therapywasunsuccessful humanin LAD-I patients, studya using foamy virus vectors canineain leukocyte adhesion deficiency model was very promising. transfusion in acute infectious episodes is debatable owing to its side effects. Patients with LAD-II (OMIM #266265) have a defect in the fucosylation of glycoproteins,various some cellof whichsurface function as selectin ligands, such as sialyl Lewis X carbohydrate(sLeX,groupsCD15a). 40 2 integrin is intact, thereby permitting β Since the genetic cause reveals that the defect 27 Defective fucosylation results in growth retardation and a In addition, the mechanisms of b2-integrin activation are patients. 36;42 II - (GDP-fucose transporter 1) at 11p11.2 (OMIM *605881), in which 7 LAD

38 FUCT1 Although recurrent bacterial infections occur in almost all patients, they are 30;36 linical stigmata in C igure 4. often not very severe and not resulting in overtas in woundLAD-I. healing Most infectionsdefects oroccur necrotic in thelesions first reportedyears atof later live, age. although periodontitis has been F 35 member C1), or different mutations have beenreported so far. Physical Examination The clinical course of LAD-II with respect LAD-I,to infectious in complications accordance is a withmilder onepatients, lowerthan the leukocyte adhesion counts. and While transmigration rolling via is defective in LAD-II apparently some neutrophil mobilization to sitesof neutrophilof defenseinflammation,and in tissues. allowing some level involves glycosylation, LAD-II has now been categorized as one of the group of the congenital disorders of glycosylation (CDG), and has been reclassified as CDG-IIc. still intact. coarse face. Long eyelashes, a broad and depressed nasal bridge, a simian crease, and dorsally positioned second toes are the clinical stigmataet al. of Leukocyte a adhesion patientdeficiency II with syndrome, LAD-II. a 1999;134(6):681–8; generalized with (From permission.) defect Marquardt in fucose T, metabolism. Brune J Pediatr T, Luhn K, 42

3 Leukocyte Adhesion Deficiencies 3 Leukocyte Adhesion Deficiencies 43

38 42 Upon 47 1,6-core α Patients are born at term, gene. The mutation seems 38;39 Expression of CR3 on LAD-II During 9 months of treatment 38;43 In addition, for one of the treated SLC35C1 48 39;43;47 One patient had coronal craniosynostosis. 38 With intravital microscopy, it was observed 36;46

39 38 The neutrophil counts remain high during childhood and then 38 44;45

49 Neutrophil values range from 6,000 to >50,000/ml in absence of infection up to 30,000 The biochemical hallmark of LAD-II is a lack of expression of fucosylated glycoconjugates, It should be mentioned that the early and late features of LAD-II, namely, moderate However,LAD-II patients present with other abnormal features, such asgrowth retardation Final proof of LAD-II arises from genetic analysis of the discontinuation of the therapy, selectin ligands again were within 7 days. lost and neutrophil counts increased patients treatment led to an autoimmune response against refucosylated antigens. Imaging and Additional Testing Complications and Concerns The metabolic pathways causing the severe unclear. Oral psychomotorfucose supplementation and may cure growthimmunological symptoms retardation in some are cases, but stilldevelopmental delay is hardly improving. Infections are commonly treated with antibiotics. In addition, high-dose oral supplementation of fucose had strong beneficial effects in some patients. Therapeutic Options & Clinical Outcome with fucose of the first patients, infections and fever disappeared, elevated neutrophil counts returned to normal, and in one However,fucose supplementation of notissuccessful allinpatients, since treatment thetwoofIsraeli patients even psychomotor Arab capabilitiespatients did not exhibit improved.a similar beneficialresponse. neutrophils is normal. withno apparent dysmorphism, but severely impaired postnatal weight gain and microcephaly reportedwere mostforpatients. somefamiliesIn intrauterine growthretardation sufficientwas to screen for LAD-II prenatally. In addition, convulsions, cerebral atrophy and autistic features were reported for more than half of the patients. fucosylated N-glycans on fibroblast proteins and blood group antigen H on erythrocytes, the latter known as the rare Bombay blood group phenotype. to 150,000/ml during infectious episodes. such as the Lewis antigens Lewis X (LeX) and sialyl Lewis X (sLeX) on leukocyteproteins,on (sLeX) X Lewissialyl and (LeX) X antigensLewisLewis the as such accompanied by neutrophilia in the first few years of life and severe mental retardation and short stature in childhood, are also prominentdisorders of glycosylationfeatures (CDGs). of other congenital (short stature), mental retardation and a coarse face (Figure 4). that LAD-II neutrophils roll poorly, only 5%, whereas the rolling fraction of control neutrophilsand LAD-I is around 30%. to determine the severity of LAD-II: whereas GFTP patients, it is is directed improperlyto the Golgi butlocated still dysfunctionalin in others,the the latter ERcorrelated with in a somemilder immunological phenotype. drop at adolescence; this finding might be explained by an improvement in adaptive immunity providingage,withbetterdefense against infections reducingstimuliandneutrophilia.thefor

27 2) is 27;56;59 β 1 α found in 3, However, β 64 A prominentA Experiments IIb 58 α 55;63 FERMT3 20;62 have been reported. FERMT3

53 , type-1/variant In addition to recurrent non-purulent III had led to the original hypothesis of kindlin-3 24;27;50-59 Based on the persistent leukocytosis, many of the 44 et al. (fermitin family homolog 3) at 11q13.1 (OMIM *607901). eficiency type So far, 11 So different far, mutations in D 3 integrin-mediated adhesion to the bone, thereby explaining the FERMT3 β 44;60 v 3). α β . Expression of integrins on neutrophils and platelets (i.e. However,since thefunctional defect suchinpatients canonlycorrectedbe However, the prevalence and manifestations of osteopetrosis differ largely dhesion 2and 60 β A 54;57;61 1, FERMT3 β Families have often lost newborns within weeks after birth, previous to the 24;53 encodeskindlin-3,protein ainvolved inside-outin signaling allbloodto cell-expressed In addition,In LAD-III patients affectedare bleeding a by tendency, similar severe more or 23 ocyte k Some patients suffering from LAD-III may also present with an osteopetrosis-like bone The molecular defect of this variant form of LAD (OMIM #612840) has since 2010 been A discussion has taken place in the literature about the importance of a genetic variation in eu integrins ( deficiency in LAD-III - confirmed as indicated by thehave subsequently description been used as ofa tool to delve variousinto LAD-III pathology. mutations - and normalslightlyor increased, integrinand activation inducedcanbeartificial by stimulation with monoclonal antibodies (mAbs) or cations. skeletal defect. by reconstitution with kindlin-3 and not by reconstitution with CalDAG-GEF1, this variationCalDAG-GEF1 in is of no importance for the functional defect in LAD-III patients. with kindlin-3 knockout mice by Moser defectadditioninincreasedthe to bleeding tendency recurrentand infections. among LAD-III patients, as unaffected bone formation is also found (Figure 5). Thereason this for heterogeneity has remained unclear. Physical Examination LAD-III patients suffer from severedelayed recurrent umbilical non-purulentcord detachment infections,with or oftenwithout precededomphalitis,LAD-I. and by leukocytosis as seen in L LAD-III, also known as LAD-1/v, was first described identifiedin1997 by in our group and about has now 20 been families worldwide. Imaging and Additional Testing As for LAD-I and -II, LAD-III should be mutationsconfirmed in by genetic analysis, in this caserevealing assigned to mutations in diagnosis of the reported patients, demonstrating the high mortality rate of LAD-III bleedingThe disorderpatients.originates platelet a from defect, indicating thatsignalingthe defect also affects the b3 integrin fibrinogen receptor aIIbb3 on blood platelets. patients were suspected to suffer from juvenile myelomonocytic (JMML). than exhibited by Glanzmann Thrombasthenia patients. infections, LAD-III patients disorder. exhibit a severe Glanzmann Thrombasthenia-like bleeding osteopetrosic phenotype was also observed in the kindlin-3 knockout mice. The cause of this osteoclasts,osteopetrosisthewithinmacrophage-likerepresentwhich liemight hematopoietic cells critical for bone resorption. Bone resorption requires the formation of a so called ‘sealing zone’ that depends on FERMT3 β thesepatients. the gene encoding CalDAG-GEF1 (a guanine nucleotide exchange factor for Rap1, involvedintegrin activation)in in some patients with LAD-III, in addition to mutations in 44

3 Leukocyte Adhesion Deficiencies 3 Leukocyte Adhesion Deficiencies 45 concentrations onlyin 2+ UZ is used to induce uptake and NADPHinduceanduptake to used is UZ 64 Vertebrae and hip joint of an osteopetrotic patient (A, patient osteopetrotic an of joint hip and Vertebrae 1), 1), thus explaining the clinically more severe bleeding β 2 α upon artificial integrin activation. 2+ patients show osteopetrosis. osteopetrosis. show patients III Platelets from Glanzmann patients are still capable of forming small aggregates aggregates small forming of capable still are patients Glanzmann from Platelets - 65 LAD ot all all ot N Many tests have been performed on LAD-III neutrophils. One example of an assay to In In addition to the recurrent infections, LAD-III patients suffer from a bleeding tendency, igure 5. 5. igure manifestations in LAD-III patients, in which all platelet integrins are functionally defective. functionally are integrins platelet all LAD-III in which in manifestations patients, upon collagen stimulation, whereas platelets from LAD-III patients are not. These require aggregates functional GPIa/IIa (integrin discriminate between LAD-I and LAD-III neutrophils is the NADPH oxidase screening test with unopsonizedgroup.describedourzymosanas (UZ)by the increased sensitivity of bone marrow (BM) or blood cellsJMML, isto negative inGM-CSF LAD-III. as the hallmark for F which which has been reported as more severe than flowcytometricaggregation bothassay, in syndromes can bediscriminated based on Glanzmann formation of Thrombasthenia. With a novel aggregates. small oxidase activity in purified neutrophils based on therequirement for kindlin-3-dependent CR3 activation before uptake of the zymosan. The response activationis andsubsequent zymosan absentuptake can beinduced highbyMg in both types of LAD, but caseLAD-III,of provingthat CR3,onceitsactivein conformation, functionalis theseinpatients. Similarly,neutrophil adhesion CR3to ligands isabsent inresponse severalto chemoattractants, but can be induced with Mn B), compared to the lumbar vertebrae and pelvis of a kindlin-3-deficient patient without osteopetrosis (C). osteopetrosis without patient kindlin-3-deficient a of pelvis and vertebrae lumbar the to compared B),

66 64 Dacrocytes (teardrop-shaped; left 2 arrowheads) and While untransplanted, the urge for transfusion differs per

mice are abnormally shaped, with striking protrusions and -/- mice. -/- kindlin-3 In addition, granulocyte transfusions have been used in at least one patient and 64 Altered levels of cytoskeletal proteins have not been reported. bnormal erythrocyte phenotype. 56 A Erythrocytes of kindlin3 Splenomegaly or hepatosplenomegaly has been observed in almost all patients, in some igure 6. are believed to increase pathogen clearance. Therapeutic Options Patients with LAD-III need prophylactic antibiotics as well as repeated blood transfusions, but the only curative therapy is HSCT. transfusionspatient50andthan20more can and riseto per yearerythrocytesfor platelets,and respectively. F only later during infancy, which expression in hematopoietic may cells, low kindlin-3 result levels have been fromreported in endothelial extramedullarycells as well, although the biological hematopoiesis. significance is unclear. Besides elliptocytes(right arrowhead) characterize the erythrocyte population ofLAD III, although the phenotype is less severe than in invaginations.cellsThehavealtered expression levelscytoskeletal of proteins,micethe andare severelyanemic. humanIn LAD-III patients, weandothers haveseen less prominent alterations of the erythrocyte population, including(Figure 6). dacrocytes (tear drop-shaped) and elliptocytes 46

3 Leukocyte Adhesion Deficiencies 3 Leukocyte Adhesion Deficiencies 47

67 ist L ledgements w no Hynes RO. Integrins:Hynes RO. bidirectional, allosteric signaling machines. Cell2002;110:673-687. Schymeinsky J, Mocsai A, Walzog mechanismsclinicaland implications. B. Thromb.Haemost. 2007;98:262-273. Neutrophil activation via beta2 DelonBrownIntegrinsNH.I, integrins actintheandcytoskeleton. Biol.Curr.Opin.Cell2007;19:43-50. (CD11/CD18): molecular Kuijpers TW, Hakkert BC, prestimulatedendothelial platelet-activatingforcells:role a Biol.factorJ.Cell1992;117:565-572. HartIL-8. and MH, Roos D. Neutrophil migration across monolayers of cytokine- Barreiro O, Sanchez-Madrid inflammatoryresponse. Rev.Esp.Cardiol. 2009;62:552-562. F. Molecular basis of leukocyte-endothelium interactions during the Zarbock A, Ley K,glycoconjugates thatmediate rollingsignalingand underBloodflow. 2011;118:6743-6751. McEver RP, Hidalgo KansasGS.Selectins A. theirand ligands: currentconcepts controversies.and Blood1996;88:3259-3287. Leukocyte ligands for endothelial selectins: specialized Murdoch C, Finn A. Chemokine receptors and 2000;95:3032-3043.their role in inflammation and infectious diseases. Blood Zimmerman GA, Prescott SM, McIntyre TM. Endothelial cell interactions with :signalingmolecules. tethering1992;13:93-100.Immunol.Today and Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascadeupdated. Nat.Rev.Immunol. 2007;7:678-689. SpringerTA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell1994;76:301-314. Hogg N, Patzak I, Willenbrock TheF. insider’s guide to leukocyte integrin signalling and function. Nat.Rev. Immunol.2011;11:416-426. CalderwoodDA, ZentR, headGrantet al.domainThe RTalin bindsintegrin to beta subunit cytoplasmic tails regulatesand integrin activation. J.Biol.Chem. 1999;274:28071-28074. Lim J, Wiedemann A, phagocytosis. TzircotisMol.Biol.Cell 2007;18:976-985. G et al. An essential role for talin during alpha(M)beta(2)-mediated Garcia-Alvarez B, de Pereda JM, Calderwood DA et al. Structural determinants of integrin recognition by talin.recognitionintegrinStructural determinantsby al.of etCalderwood JM, PeredaDA de Garcia-Alvarez B, Mol.Cell2003;11:49-58. Shattil SJ, Kim C, Ginsberg MH. The final steps of integrin activation: the end2010;11:288-300. game. Nat.Rev.Mol.Cell Biol. k

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eference

Complications and Concerns Whereas the success rate of HSCT is improving over the last andthebleeding disorderyears,often cause major complications thetreatmentpre-transplantin LAD-IIIof patients. infections In addition, osteopetrosis may complicate the conditioning regimen. Clinical Outcomes The survival of untransplanted LAD-IIIdemonstratedthe incidenceby deceasedof siblings whowerenotdiagnosed butsuffered from patients is low, and similar symptoms. the Less than 4 highpatients have so mortalityfar survived childhood is without HSCT, oldestand furtherreportedthe patient has reached the age of 20, although the need for platelet transfusions has increased to transfusions1-2 per week (unpublished data). Upon successful HSCT, patients may continue live without further symptoms. 8. 9. 10. 11. 7. 5. 6. 3. 4. 2. R 1. A We gratefully acknowledge Prof. Dirk Roos for critically reading the manuscript. 12. 13. 14. 15. 16. Han J, Lim CJ, Watanabe N et al. 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