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Leukocyte functions disorder
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- 2. Introduction • Essential cellularcomponents of blood. • Develop from the pluripotential hematopoietic stem cells in the bone marrow. • Leukocytes include Granulocytes: – Neutrophils – Basophils – Eosinophils Monocyte Lymphocyte
- 3. Primary function: Toprotect the host from infectious agents or pathogens. Innate Immune Response Acquired/Adaptive /Specific immune system • 1st response to common class of pathogens. • Neutrophils & Monocytes – Play a major role. • Rapid but limited. • Initiated in lymphoid tissue when pathogens encounter lymphocytes. • Lymphocytes – plays a major role.
- 4. Neutrophils • Neutrophils arevery mobile cells • Two types of granules: – Primary or azurophilic – Secondary or specific • All granules contain enzymes which are involved in killing and digesting bacteria and fungi.
- 5. Functions of Neutrophils •Innate immune response: 4 steps 1. Adherence 2. Migration ( chemotaxis) 3. Phagocytosis 4. Bacterial killing
- 6. 1. Adherence • Activationof leukocytes & vascular endothelial cells (VEC) by inflammatory mediators(cytokines). • Activation result is activity of 3 classes of cytoadhesion mol. (CAM) & their ligands. • CAM & their ligands play a major role. 1. Selectins & their ligands 2. Intercellular adhesion molecule (ICAM) 3. Beta 2 (CD18) family of leukocyte integrin & their ligands.
- 8. 2. Migration- pseudopodiaforms ICAM E & P Selectins
- 9. 3. Phagocytosis Some pathogensare recognized by opsonization (IgG & C3b) PAMP PRR Degranulation
- 10. 4.Bacterial Killing &Digestion • Phagosomes fuse with the lysosome( phagolysosome) discharge their granules Bacterial killing & digestion. • Microbicidal mechanism – 2 types 1. Oxygen dependent 2. Oxygen independent
- 11. Oxygen Dependent • Neutrophilsactivation is accompanied by increase in O2 use- Respiratory burst/ oxidative burst. • Respiratory burst – caused by activated NADPH oxidase. Increase in O2 consumption. Increase glycolysis. Production of ROS ( superoxide) Increase in glucose oxidation by HMP shunt • Activated NADPH – detected by NBT, Cytochrome oxidase or chemiluminescence test.
- 12. • MPO independent: H2O2 Superoxide ion Hydroxyl ion Singlet oxygen • MPO dependent: Present in primary granules
- 13. Oxygen Independent • Mediatedby the granules present in primary,gelatinase,specific granules. • Acid pH of phagosomes Increase activity of granular protein Microbicidal activity • Alone insufficient to kill microbes. • Include : Lysozyme Lactoferrin BPI( Bacterial permeability inducing proteins) Defensins Collagenase
- 14. Monocytes Functions • Activein both innate and adaptive IR. • Phagocytosis. • Ingest activated clotting factors thus limiting the coagulation process. • Secrete a variety of substance affects the functions of other cells especially lymphocytes.
- 15. Eosinophils • Eosinophils :2 - 8 % leukocytes • Derived from hematopoietic stem cells • bilobed nucleus with numerous bright orange cytoplasmic granules • Originates from IL -5 responsive CD 34 + myeloid progenitor cells • IL -5 has lineage specificity for eosinophils
- 16. Function of Eosinophils •Cationic proteins, cytokines and leukotriens- in eosinophil granules mediate Parasite defense Allergic response Tissue infiltration Immune modulation
- 17. Basophils • Basophils areleast of the circulating leucocytes • They constitute less than 1% of the circulating leucocytes or absolute counts of 0.02-0.08x109/lt • Normal range on BMA is 0-0.5% • Circulates in blood and normally not found in tissues • Recruited to tissues during inflammatory or immunologic response
- 18. Functions of Basophils •Basophils – Key player in Allergy • Degranulation • Late Phase Reaction
- 19. Lymphocytes • Specific componentof immune system. • Different types : B-Lymphocytes (“B cells) T- Lymphocytes (“T cells”) Natural Killer Cells (NK cells) Memory Cells Suppressor Cells • They have different functions in specific immunity.
- 20. B-LYMPHOCYTES • Mature inbone marrow, then carried to lymphoid tissue via blood stream and lymphatic circulation. • This process of maturation and migration takes place throughout life. • Other lymphocytes can be generated via mitosis of B lymphocytes resident in lymphoid tissues
- 21. T-LYMPHOCYTES • Immature lymphocytesleave bone marrow during fetal and early neonatal life. • Go to thymus gland. • Mature there before they go on to other lymphoid tissues.
- 22. Role in SpecificImmunity • The body must be able to recognize the difference between “self” and “nonself.” • Any lymphocytes with antibodies that recognize one’s own body tissue as an antigen are killed during fetal life. • Stages of specific immune response- 1) Antigen encounter and recognition by lymphocytes. 2) Lymphocyte activation. 3) Attack
- 23. 1) Antigen encounterand recognition by lymphocytes • Specific lymphocytes are programmed to recognize a specific antigen. • Usually happens in a lymphoid organ, bloodstream, or lymph vessel. 2) Lymphocyte activation: • Once a lymphocyte has recognized an antigen, it undergoes numerous cycles of mitotic divisions, making more of the same. • Some of the newly produced cells carry out the attack; others influence the activation and function of the attack cells
- 24. 3) Attack: B-lymphocytes havespecific receptors on their cell membrane – ANTIBODIES – that bind with invading materials/organisms
- 25. Function of TLymphocytes • Do not produce antibodies. • Function in “cell-mediated immunity.” • “NATURAL KILLER” cells destroy viruses. • Secrete “lymphokines” which attract phagocytic cells. • Secrete “perforin” which eats the cells membrane or viral coat of invaders.
- 26. • Helper Tcells: • Induce macrophages to destroy other antigens • STIMULATE B-LYMPHOCYTES TO PRODUCE ANTIBODIES. • “Suppressor T Cells” prevent overreaction of the system. (Inhibit B-lymphocye production.)
- 27. Disorders of LeukocyteFunction 1. Disorders of opsonisation and ingestion 2. Defective adhesion of Leukocytes. a) Leukocyte adhesion deficiency b) Drug induced 3. Defective locomotion and chemotaxis a) Actin polymerization abnormalities. b) Neonatal neutrophils c) Interleukin 2 administration d) Cardiopulmonary bypass e) Enhanced motile response
- 28. 4. Defective microbialkilling a) Chronic granulomatous disease b) RAC – 2 deficiency c) Myeloperoxidase deficiency d) Hyper immunoglobulin syndrome e) G6PD deficiency f) Extensive burns g) Glycogen storage disease h) Ethanol toxicity i) End stage renal disease j) Diabetes mellitus
- 29. 5. Abnormal structureof the nucleus or of an organelle a) Hereditary macropolycytes b) Hereditary hyper segmentation c) Pelger- Huet anomaly d) Alder- Reilly anomaly e) May – Hegglin anomaly 6. Degranulation defect a) Chediak – Higashi disease b) Specific granule deficiency
- 30. Disorders of opsonisationand ingestion Basic Pathogenesis Defect Impaired Function Clinical consequence • Synergistic action of Ig & complement creates opsonins •Coats microorganism • Stimulate chemotaxis •Def impaired Fn • Ig & C3 deficiencies • Properdin deficiency • Mannose binding protein def. Deficiency of chemotaxis & opsonins activity Recurrent pyogenic infections
- 31. Defective Adhesion Main functionaldefect • Unable to successfully activate and "grab" or adhere to the blood vessel wall because they lack the necessary podium & if present but may be defective and not work properly. • Since these leukocytes cannot adhere, experience an "adhesion deficiency",
- 32. Leukocyte Adhesion deficiency Dis order Inherit ance DefectGenetic defect Impaired Function Clinical course LAD – 1 AR Absence of CD11/CD18 surface adhesive GP( B integrin) on leukocyte membrane Failure to express CD 18 m RNA • Rolling normal •Decreased binding of C3bi to leukocytes •Impaired adhesion to ICAM 1 & 2. • Neutrophilia • Recurrent bacterial infection. • Delayed umbilical cord separation LAD – 2 AR Loss of fucosylation of ligands for selectin ( Cd15s expression) Mutation of the GDP fucose transporter • Donot roll well. •Decreased adhesion • Neutrophilia • Recurrent bacterial infection without pus. LAD – 3 AR Impaired integrin functions. Defective kindlin – 3 , binds to B- integrin -> integrin activation Mutation of FERMT 3- encodes kindlin- 3 • Impaired leukocyte adhesion & platelet activation • Neutropenia • Recurrent infections. • Bleeding tendency
- 33. Disorder of CellMotility Disorder Inherit ance Defect Genetic defect Impaired function Clinical course 1.Enhanced motile response ( familial Mediterranean fever) AR Defective Pyrin regulation of caspase 1 & thereby IL-B secretion. Mutation a gene encodes pyrin( chromosome – 16) • Increase sensitivity to endotoxin. • Excessive accumulation of leukocytes. • Recurrent fever. •Peritonitis. •Pleuritis. • Arthritis • Amyloidosis 2.Immue complexes • Rheumatoid arthritis. • SLE & other inflammatory condition. •Binds to Fc receptors on neutrophils. • Impaired chemotaxis Recurrent pyogenic infections 3. Actin polymerization abnormality • Actin doesn’t polymerize well. • Lower level of F-actin. Over expression of Leufactin ( lymphocyte specific protein 1)- F actin binding Defect in chemotaxis, adhesion and phagocytosis Recurrent bacterial infections
- 34. Defective microbial killing •The ultimate step in the elimination of infectious agents and necrotic cells is their killing and degradation within neutrophils and macrophages. • Any defect or deficiency causes recurrent infections
- 35. Chronic Granulomatous Disease •Affecting the functions of neutrophils & monocytes. • Defect : Absent or Reduced function of the respiratory burst. • Genetic defect: Congenital defects in the five components of the enzyme NADPH oxidase. • Clinical features: Rucurrent catalase +ve infections • Hallmark : presence of granulomas caused by thechronic inflammatory response to the pathogen
- 36. • Functional defects:Defects in opsonisation, phagocytosis, or intracellular killing. • Lab diagnosis: 1. Bacterial killing test 2. Phorbol myristate acetate (PMA) 3. Nitroblue tetrazolium (NBT) test 4. Flow cytometric analysis (Most sensitive) 5. Prenatal diagnosis of CGD :Analysis of DNA from chorionic villous sampling or amniotic fluid cells
- 37. Defective Microbial Killing DisorderInheri tance Defect Genetic defect Impaired functions Clinical course 1. G6PD Deficiency < 5 % of normal activity of G6PD • Failure to activate NADPH oxidase( required for phagocytosis). • Hemolytic anemia Infection with catalase +ve organism 2. RAC – 2 Deficiency AD Negative inhibition by mutated protein of Rac- 2 mediated function Mutation in Rac- 2 Failure to activate receptor mediated O2 generation & chemotaxis. • Neutrophilia. • Recurrent bacterial infections 3. Deficiencies of glutathione reductase & synthetase AR Due to def. Failure to detoxify H2O2 Excessive formation of H2O2 Minimal problem with recurrent pyogenic infections
- 38. Defective Microbial Killing DisorderInherit ance Defect Genetic defect Impaired functions Clinical course 4. Hyperimmuno globulin – E syndrome ( Job syndrome) • AD • Spora dic • Defective STAT 3 protein ( major transductio n protein) Missense mutation or in-frame deletion in STAT 3 protein • Poor Ab & cell mediated responses. • Impaired chemotaxis. •Impaired regulation of cytokine productions. • Recurrent skin & sinopulmonary infection • Retained Pri teeth • Facies characteristic • Blood & sputum eosinophilia. 5. MPO deficiency AR Absence of MPO In N. & M Failure to process modified precursor protein due to missense mutation H2O2 dependent antimicrobial activity not potentiated by MPO.
- 39. Degranulation Abnormalities • Normallyin stimulated cells signal transduction cascade activates G proteins enhanced intracellular Ca2+ , protein kinase activation. • Culminate in secretion ( Fusion of granule membrane with phagosomes of the plasma membrane) – Degranulation. • Defect in degranulation- Reduce bactericidal activity ( Defective Leukocytic function)
- 40. Chediak- Higashi Syndrome •Congenital gigantism of peroxidase positive granules. • Inheritance – AR • Basic defect : Increased fusion of cytoplasmic granules Abnormal large peroxidase positive granules. • Giant granules are formed in most granules containing cells through out the body. • Genetic defect: CHS1/LYST gene –encodes a protein , regulate granule fusion.
- 41. Pathogenesis: Early stage ofMyelopoiesis Azurophilic granules fuse to form giant granules Large granules- Reduced hydrolytic enzymes Precursors - phagocytosed in the marrow Moderate Neutropenia In PB- Normal ingestion of particles & O2 met. By Leukocyte Slow & inconsistence delivery of hydrolytic enzymes Slow Microbial killing Bacterial Infection CHS Cell Mb.- More fluid than normal. 1. Fusion of granules. 2. Reducing expression of CD11b/CD18(M ac-1) 3. Increase C-AMP Decrease chemotaxis
- 42. • Monocytes –same functional derangement. • Perforin deficient NK cells impair cytotoxic activity unable to kill microbes.
- 44. Clinical Course: • Characteristicallypresent with partial albinism , silvery hair & photophobia. • Recurrent pyogenic infections. • Increase bleeding tendencies. • Usually quiescent. • >85% of patients Accelerated phase Lymphadenopathy, neutropenia,hepatosplenomegaly as a manifestation of hemophagocytic syndrome.
- 45. Lab Findings: • Characteristicmicroscopic findings 1. Large, often multiple, peroxidase +ve granules in the granulocyte of blood and BM. 2. Large melanosomes in the hair. • Abnormal platelet aggregation (def. of storage pool of ADP & serotonin). • Early phase- blood counts normal • Late stage – cytopenias.
- 46. Specific Granules Deficiency InheritanceFunctional defect Genetic defect Impaired Function Lab . Findings • AR • Acquired form Thermally injured Pt. MDS • Absence of specific granules (ALP+) • MPO +ve primary granules present. • Confined to myeloid cells only. • Functional loss of myeloid transcription factor due to Mutation or reduced expression of growth factor independence (Gfi) and C/EBPE which regulates specific granules formation • Impaired chemotaxis & bactericidal activity. • Bilobed nuclei in neutrophils. •Def. of Defensin,gelatina se, collgenase, vit B12 binding protein & lectoferrin. (Present in sec granules.) • Presence of neutrophils devoid of specific granules but containing azurophilic granules on PB. • Confirmatory test- def. Of lactoferrin or vit B12 binding protein.
- 47. Abnormal Structure ofthe Nucleus or Organelle Disorder Morphological defect Impaired function Clinical course or associated condition 1. Pelger – Huet Anomaly • Distinct shapes of the nuclei of leukocyte with decrease number of nuclear segment. • Nuclei –Rod like , dumb bell shaped, pinch nez with small, round or oval individual lobes. Function normally 2. Alder – Reilly Anomaly • Large, dark azurophilic & basophilic granules in all leukocytes. •Distinguish from toxic granules by staining metachrmoatically with toluidine blue. • Lymphocytes – occur in clusters surrounded by vacuoles & shaped like a dot or comma ( Gasser’s cell) Associated with Mucopolysaccharidosis
- 48. Abnormal Structure ofthe Nucleus or Organelle Disorder Morphological Defect Impaired Functions Clinical course / associated conditions 3. May Hegglin Anomaly ( Autosomal dominant) •Giant fused , well defined granules in granulocytes & lymphocytes. • Inclusion similar to Dohle bodies – distinguished from it as these are more large & rounded. Engulf but donot kill microorganism. • Serious, often fatal. • Repeated pyogenic infections 4. Hereditary Hypersegmentation ( Autosomal dominant) Increase number of neutrophilc segments 5. Hereditary hypersegmentation of eosinophils and –ve staining for peroxidase Lack of sudanophilia & peroxidase activity
- 49. Basophilic inclusions May Hegglinanomaly Note the granules Alder Reilly anomaly- hurler’s syndrome Pelger Huet Anomaly
- 50. Workup of thePatient with Recurrent Infections
- 51. 1..Initial evaluation • History,physical examination. • Family history • Blood counts •Bacterial counts Functional asplenia Wiskot- Aldrich syndrome Neutropenia work up ( BM,ANA,CBC) Howell Jolly Bodies Thrombocytopenia, eczema Neutrophil <1500 Neutrophil G6PD def. Chediak – Higashi syndrome Specific granules Def. Hemolytic anemia Partial albinism, abnormal granules Abnormal granules Pelger-Huet anomaly If normal •2. Ig/Complement work up- Ig Levels. •Ab titers • Delayed hypersensitivity •Total T cells Hypogammaglobulin syndromes e.g. linked agammaglobulinemia Severe combined immuno def, AIDS, Mucocutaneous candidiasis,ataxia- telegiectasia Hypocomplementemia syndrome IgE.- 2000 IU/ml Hyper- IgE Decreased Ig Cellular Immuno def. Decreased complement Stat 3 mutation If normal 3. Phagocyte evaluation. •NBT •DHR assay • Chemotaxis assay If normal Neutrophil G6PD def. LAD.Chediak-Higashi syndrome, Specific granules Def,Rac-2 def. Chronic granulomatous ds Complement def, humoral defects Absent O2-, Abnormal NBT,DHR Only abnormal chemotaxis Abbreviated O2- production - GSH pathway Abnormal response to activated control serum 4.Further phagocyte evaluation. • MPO stain •Flow cytometry(CD11/18) •Quantitative ingestion assay MPO Def Opsonins Defect Neutrophil actin dysfn MPO absent Decrease ingestion with Pt’s serum Decreases ingestion with control serum LAD-1. LAD-2 LAD-3. Absent Cd11/18 Decreased sLe expression by flow Failure to generate O2- when challenged with unopsonized zymosam
- 52. References • Wintrobes clinicalhematology • William’s hematology • Postgraduate hematology • McKenzie Clinical lab hematology • Robbins basic pathology
- 53.
Editor's Notes
- #15 If granulocyte activation persists,neutrophils release substances such asmonocytic chemotactic protein that attract monocytes to the area. These in turn release monokines that enhance lymphocytic infiltration. Monocytes phagoctosed cellular debris , effete cells &n other particulate matter.
- #32 leukocytes are unable to successfully activate and "grab" or adhere to the blood vessel wall because they lack the necessary "hooks". Sometimes the "hooks" are present but may be defective and not work properly. Since these leukocytes cannot adhere, because of missing or defective "hooks", they experience an "adhesion deficiency“,
- #33 LAD 2- CD 18 expression is normal/selectin and their ligand ( carbohydrate whose structure is related to sialyl lex (CD 15s) – Important role in neutrophils adhesion so lack of Cd 15 s expression didn’t bind to surface. The Kindlin-1, -2, and -3, perform an essential role in activation of integrin adhesion receptors,
- #34 Pyrin is produced in certain white blood cells (neutrophils, eosinophils and monocytes) that play a role in inflammation and in fighting infection. pyrin helps regulate inflammation by interacting with the cytoskeleton. Pyrin may direct the migration of white blood cells to sites of inflammation
- #36 X linked recessive disease caused by a mutation in the glycoprotein 91phagocyte oxidase gene (gp91 phox gene), which encodes a membrane bound subunit of the enzyme; the remaining one third of cases are inherited in an autosomal recessive manner and are caused by defects in the cytosol components p47phox (25%), p67phox (5%),or the smaller membrane bound subunit,p22phox (5%). In general, patients with p22phox and p67phox deficiency CGD have a similar clinical phenotype to those with X linked 91phox deficiency, but patients with 47 phox deficiency follow a milder course.85–88 Patients with X linked CGD can, on the basis of genotypic analysis, be divided into the X910 (complete eficiency), X91− (partial deficiency), or X91+ (stable but inactive gp91 phox) subgroups.89
- #37 The bacterial killing test is a comprehensive screening test for defects in opsonisation,phagocytosis, or intracellular killing. It looks at the ability of phagocytes to kill catalase positive bacteria, The phorbol myristate acetate (PMA) nitroblue tetrazolium (NBT) slide test is an extremely easy and reliable screening test for disorders of oxidative metabolism 3. Neutrophils are exposed to a stimulus,such as PMA, incubated with NBT, and staining assessed by counting 100 neutrophils on a smear. After stimulation, more than 95% of neutrophils from healthy individuals stain positive, with a blue black precipitate of formazan granules, whereas < 5% of neutrophils are stained in individuals with CGD. Carriers of X linked CGD usually show 30–70% positive cells Flow cytometry:Neutrophils are stimulated in vitro using reagents such as PMA and the superoxide or hydrogen peroxide generated is measured.
- #38 Rac proteins are members of the Rho family of GTPases and are key mediators of phagocyte functions, through their involvement in the control of migration to the site of infection, phagocytosis and reactive oxygen species (ROS) production by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Rac-GTP is a component of the membrane-assembled NADPH oxidase complex,
- #39 Stat 3 is a major transduction protein affecting pathways involving would healing. Angiogenesis, immunity and cancer. Mpo Catalyses the production of HOCL in phagosome Microbicidal def if leukocyte early after ingestion of microorganism.
- #45 Accelerated phase due to lyphocytic infiltration in liver, spleen, arrow & CNS.
- #47 Impaired chemotaxis is due to def of intracellular pool of molecules exhibit in the tertiary or sec granules.
- #52 The flow cytometric dihydrorhodamine 123 (DHR) assay -The DHR assay measured change in fluorescence of DHR-loaded granulocytes after phorbol myristate acetate (PMA) stimulation. . Chemotaxis assay is based on the premise of creating a gradient of the chemotactic agent and allowing cells to migrate through a membrane towards the chemotactic agent. If the agent is not chemotactic for the cell, then the majority of the cells will remain on the membrane. If the agent is chemotactic, then the cells will migrate through the membrane and settle on the bottom of the well of the chemotaxis plate.