The Beloved : Its Function in Health and Disease Multipotent Progenitor

Myeloid Lymphoid CMP IL-3, SCF, GM-CSF CLP Committed Progenitor MEP GMP

GM-CSF, IL-3, SCF EPO TPO

G-CSF M-CSF IL-5 IL-3 SCF RBC Platelet Neutrophil / Basophil B-cells Eosinophil T-Cells Mast cell NK cells Mature Cell

Dendritic cells PRODUCTION AND KINETICS OF

CELLS % CELLS TIME Marrow: Myeloblast 1 7 - 9 Mitotic 4 Days 16

Maturation/ Metamyelocyte 22 3 – 7 Storage Band 30 Days Seg 21

Vascular: Peripheral Seg 2 6 – 12 hours 3 Marginating Pool and ? Tissue clearance by 0 – 3 days PHAGOCYTOSIS

1. Mobilization 2. 3. Recognition (Opsonization) 4. Ingestion 5. Degranulation 6. Peroxidation 7. Killing and Digestion 8. Net formation

Adhesion: β 2 Integrins ▪ Heterodimer of and chain ▪ Tight adhesion, migration, ingestion, co- stimulation of other PMN responses

LFA-1 Mac-1 (CR3) p150,95 22 CD11a CD11b CD11c CD11d CD18 CD18 CD18 CD18 Cells All PMN, Dendritic Mac, mono, leukocytes mono/mac, PMN, T cell LGL Ligands ICAMs ICAM-1 C3bi, ICAM-3, C3bi other other Fibrinogen other

GRANULOCYTE CHEMOATTRACTANTS

Chemoattractants Source Activators Lipids PAF Neutrophils C5a, LPS, FMLP Endothelium

LTB4 Neutrophils FMLP, C5a, LPS Chemokines () IL-8 , endothelium LPS, IL-1, TNF, IL-3 other cells Gro , , Monocytes, endothelium IL-1, TNF other cells NAP-2 Activated platelets Platelet activation Others FMLP C5a Activation of complement

Other Important Receptors on PMNs

Pattern recognition receptors – Detect microbes Toll receptor family Mannose receptor Glucan receptor – fungal cell walls Cytokine receptors – enhance PMN function G-CSF, GM-CSF TNF Receptor Opsonin receptors – trigger phagocytosis FcRI, II, III Complement receptors – Mac1/CR3 (CD11b/CD18) – C3bi CR-1 – C3b, C4b, C3bi, C1q, Mannose binding

From JG Hirsch, J Exp Med 116:827, 1962, with permission. CONTENTS OF NEUTROPHIL GRANULES

I. Azurophil granules Functions Acid Hydrolases (glycosidases) Degradation of ingested material

Neutral proteases (cathespin G, Destruction of inflamed tissue )

Lysozyme Digestion of bacterial cell wall

Defensins and bactericidal/ Oxygen-independent bacterial killing

Myeloperoxidase Oxygen-dependent bacterial killing

CONTENTS OF NEUTROPHIL GRANULES

II. Specific granules Functions Lysozyme Digestion of bacterial cell wall Cobalamin-binding proteins Binding of cobalamin analogues Apolactoferrin Binding of free iron Collagenase Digestion of connective tissue C5a-splitting enzyme Release of C5a Heparinase Digestion of connective tissue Laminin and thrombospondin Adhesion to basement receptors membrane CD11/18 (C3bi) receptor Adhesion to ICAM-1, 2, 3 on endothelium and phagocytosis of C3bi coated particles Cytochrome B558 Component of NADPH

Infectious complications of phagocyte disorders (either quantitative or functional) Frequent and/or unusually severe bacterial, fungal - Skin, lymph nodes, lungs (portals of entry); other sites via bloodstream or tissue extension Unusual site - e.g. liver or brain abscess Recurrent/chronic , aphthous ulcers Staphylococcal common. Also Strep; Gram- negatives, unusual or opportunistic pathogens e.g. Aspergillus, Serratia, B. cepacia, Klebsiella, atypical Mtb

Inherited Defects in Neutrophil Functions

Adhesion: Chemotaxis: Leukocyte Adhesion Deficiencies (LAD) I, II, III LADI HyperIgE Syndrome Chediak-Higashi Phagocytosis: Actin defects LADI Killing: Chediak-Higashi Chronic granulomatous disease Specific granule deficiency Chediak-Higashi MPO deficiency (with DM)

LEUKOCYTE ADHESION DEFICIENCY In Vitro Leukocyte Functional Abnormalities

Clinical Features

Pediatric age group Delayed umbilical cord separation Persistent Defective neutrophil mobilization (reduced formation) Impaired wound healing Recurrent (life-threatening) bacterial and sometimes viral infections PMN/Mo adherence and spreading PMN aggregation Lekstrom-Himes JA, Gallin JI. diseases caused by defects in phagocytes. N Eng J Med 343:1703,2000. Leukocyte Adhesion Deficiency I (LAD I)

Prevalence Rare Age Usually presents in infancy Genetics AR in CD18 subunit of 2 integrin, leading to complete or partial loss of 2 integrin expression Pathogenesis Defects in adhesion, migration, phagocytosis Clinical Omphalitis, periodontitis, skin/soft tissue infections, , , poor wound healing, delayed cord separation. Staph, gram negatives Labs , absent CD11/CD18 (including Mac-1) Management HSCT, otherwise supportive care SUMMARY OF CGD

Incidence: 4 per million births

Infections: Pneumonia (70%) Aspergillus Supportive Adenitis (53%) Staphylococcus Subcutaneous Abscess (42%) Staphylococcus Liver Abscesses (27%) Staphylococcus Osteomyelitis (25%) Serratia Sepsis (18%) Salmonella

INFECTIONS IN CGD

Pathogen Presentations

Bacterial

Staphylococcus aureus Soft tissue , Lymphadenitis, Liver Abscess, Osteomyelitis, Pneumonia, Sepsis

Burkholderia Pneumonia, Sepsis

Serratia marcescans Pneumonia, Osteomyelitis, Sepsis, Soft tissue infection

Nocardia species Pneumonia, Osteomyelitis, Brain abscess Fungal

Aspergillus species Pneumonia, Osteomyelitis, Brain abscess

Candida species Sepsis, Soft tissue infection, Liver abscess

Examples of Infectious Complications of CGD Impetigo

Lymphadenitis

Aspergillus Lekstrom-Himes and Gallin, NEJM 343, 1703, 2000. 2000 pneumonia Massachusetts Medical Society. All rights reserved.

Pathology: Lung resection for Silver stain: Chronic inflammatory persistent Aspergillus Aspergillus infiltrate, foci of hyphae neutrophils

SUMMARY OF CGD

Other complications: Gastric Outlet Obstruction (15%) Urinary Tract Obstruction (10%) Colitis (17%)

Cause of Death: Pneumonia/Sepsis, Aspergillus B cepacia

NET C

Yost, C., Cody, M., Harris, E., Thornton, N., McInturff, A., Martinez, M., Chandler, N., Rodesch, C., Albertine, K., Petti, C., Weyrich, A., & Zimmerman, G. (2009). Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates. Blood. 113 (25), 6419-6427. Chronic Granulomatous Disease (CGD)

Pathogenesis Impaired microbial killing due to deficient production of oxidants Clinical a) Lymphadenitis, skin infections, pneumonia, hepatic abscess, pneumonia b) Aspergillus, B. cepacia most problematic; Staph most common. Gram-negative (eg. Serratia). Pathogens often opportunistic or unusual. However, can kill catalase-negative organisms (eg. Strep) using microbial H2O2. c) Granulomatous , including GI tract Labs Absent or markedly reduced NADPH oxidase activity Management Prophylactic trimethoprim/sulfa, itraconazole, IFN; Aggressive/prolonged treatment of infections; Prednisone/ immunisuppr for inflammation

• Definition: Reduction in the absolute neutrophil count (includes bands and segmented PMNs) below norms for age and ethnic groups in the blood circulation. • Age-related ANC: Term newborn (1 week): < 1500 Infant (1 month – 4 years): < 1000 Child, adolescent, adult: < 1500 • Ethnicity: < 800 Clinical Risk Assessment • None: ANC of 1,000 to 1,500/µL

• Moderate: ANC of 500 to 999/µL

• Severe: ANC of 300 to 499/µL

• Very severe: ANC of < 300/µL

Clinical Risk Assessment

• Acute vs. chronic lasting more than three months (ANC < 500/µL).

• Can neutrophils be mobilized from ?

• Production vs. destruction.

Epidemiology

• Acute neutropenia occurs frequently.

• Congenital neutropenia: ~ 2 per million.

• : ~ 0.6 per million. Suspicion of Neutropenia

• Acute severe bacterial infections.

• History of recurrent infections.

• Prolonged or elevated temperature (> 101° F).

• Pneumonia, , GU tract infection, buccal and tongue ulcers, chronic gingivitis, cellulitis, perirectal infections.

• Findings associated with a malignancy, immunodeficiency syndrome, viral infection or drug exposure. Laboratory Evaluation of Neutropenia

• CBCPD and blood smear. • If neutropenia is recurrent, repeat CBCPD 3x/week for 6 weeks. • Coombs test. • Immunoglobulin levels and lymphocyte subsets. • Antineutrophil antibodies. • Serology for viral infections if acute process. • ANA, LDH, uric acid. • Bone marrow exam and cytogenetics. Human Neutrophil Alloantigens

Clinical Significance Frequency Allele Frequency (%) Africans+ (%) Whites Previous Allele Frequency Antigens Names Carrier Glycoproteins (%) Asians* HNA-1a NA1 FcȣR IIIb (CD16) 88-91 46-66 57-62 AIN, ANN, TRALI

HNA-1b NA2 FcȣR IIIb (CD16) 51-54 78-84 88-89 AIN, ANN, TRALI

HNA-1c SH FcȣR IIIb (CD16) < 1 23-31 5 AIN, ANN, TRALI

HNA-2 NB1 58-64 kDa (CD177) 89-99 98 87-97 AIN, ANN, TRALI, febrile transfusion reaction, drug-induced neutropenia

HNA-3 5b CTL2 (Unknown) nt nt 89-96 ANN, TRALI, febrile transfusion reaction

HNA-4 HART CR3 (CD11b) nt nt 99 AIN, ANN

HNA-5 OND LFA-1 (CD11a) 81 88 86-92 unknown Severe Chronic Neutropenia • Heterogeneous group of disorders of myelopoiesis

• Associated with - decreased production of neutrophils - recurrent bacterial infections

• Severity of disease related to degree of neutropenia

Severe Congenital Neutropenia (SCN)

• Early childhood onset with ANC < 200

• Bone marrow shows maturation arrest at promyelocyte-myelocyte stage

• Recurrent life-threatening bacterial infections

• Associated with ELANE (ELA2)

Clinical Issues in SCN

• 10% – 30% risk of evolving into MDS/AML

• HCT should be considered in G-CSF non responders to • > 20µg/kg/day or converting to MDS/AML

• Need for annual bone marrow to survey for cytogenetic changes

• Patients remain at risk for infection because of impaired neutrophil function

Cyclic Neutropenia

• Dominantly inherited • Cycle of neutropenia q 21 days • Marrow during neutropenia: myelocyte arrest • Stem cell regulatory defect

Cyclic Neutropenia

• Like autosomal dominant SCN, cyclic neutropenia has been linked to mutations in • ELANE (ELA2) mutations found in essentially 90% of cyclic neutropenia • NOT associated with an increased risk of AML Dynamics of the Absolute Monocyte Count

Congenital Neutropenia Cyclic Neutropenia 6000 6000

5000 5000

4000 4000

3000 3000

2000 2000

Peripheral Leukocyte Count (counts/µl) Count Leukocyte Peripheral 1000 1000

0 0 0 21 42 63 84 105 126 0 21 42 63 84 105 126 Days ANC ANCAMC AMC Table 3: Classification of Congenital Neutropenia Disorders

Main hematologic Other clinical Disease (inheritance) features features (chromosomal location)

Disorders of Myelopoiesis -Cyclic neutropenia (AD) Periodic neutropenia None ELANE (19q13.3) -Severe congenital neutropenia (AD) Neutropenia, MDS/AML None ELANE (19q13.3) -Severe congenital neutropenia (AD) Neutropenia, MDS/AML None Gfi1 (1p22) -Severe congenital neutropenia (AD) Neutropenia None G-CSFR (1p35 34.3) -Severe congenital neutropenia (AR) Neutropenia, MDS/AML Neurologic HAX1 (1q21.3) or Kostmann disease impairment

Disorders of ribosomal and dysfunction -Shwachman-Diamond (AR) Neutropenia Exocrine SBDS (7q11.22) Aplastic deficiency MDS/AML Metaphyseal dysostosis

-Dyskeratosis congenita (XLR) Abnormal skin pigmentation, DKC1(Xq28) -Dyskeratosis congenita (AD) MDS/AML dystrophy, TERC (3q26)* -Dyskeratosis congenital (AR) oral , TERT (5p33)* epiphora, pulmonary fibrosis, TINF2 (14q11.2) short stature, hair loss, NOP10 (15q14-q15) developmental delay, NHP2 (5q35.3) of TCAB1(17q13.1) head and neck

Disorders of Metabolism -Reticular dysgenesis (AR) Neutropenia Sensorineural hearing loss AK2 (1p31-p34) Lymphopenia

- (XLR) Neutropenia Cardiomyopathy TAZ1 (Xq28)

-Glycogen storage disease type 1b (AR) Neutropenia Hypoglycemia, G6PT1 (11q23) hyperlipidemia, hyperuricemia, growth retardation, , renal hypertrophy

-Glucose-6-phosphatase Neutropenia Cardiac abnormality, G6PC3 (17q21) catalytic subunit 3 (AR) prominent superficial venous pattern, hepatosplenomegaly, cryptorchidism,

-Pearson syndrome (mitochondrial) Neutropenia Vacuolization of erythroid Mitochondrial DNA Pancytopenia and myeloid precursors deletion Ringed sideroblasts Table 3: Classification of Congenital Neutropenia Disorders Cont.

Main hematologic Other clinical Gene Disease (inheritance) features features (chromosomal location)

Disorders of vesicular transport -Chediak-Higashi syndrome (AR) Neutropenia Pigmentary dilution affecting LYST/CHS (1q42.1- Platelet and NK cell hair, skin and ocular fundus, q42.2) dysfunction risk for hemophagocytic syndrome

-Cohen syndrome (AR) Neutropenia Developmental delay, facial COH1 (8q22-q23) dysmorphism, retinitis pigmentosa

-Griselli syndrome type II (AR) Pancytopenia Pigmentary dilution of the skin 27A (15q14.1) and hair

-Hermansky-Pudlak type II (AR) Neutropenia AP3B1 (5q14.1)

-p14 deficiency (AR) Neutropenia, Short stature, MAPBPIP (1q21) Decreased B&T lymphocytes, hypogammaglobulinemia

Disorders of Immune Function -Cartilage-hair hypoplasia (AR) Neutropenia Short-limbed dwarfism, RMRP (9p21-p12) Lymphopenia fine hair, immodeficiency, Macrocytic anemia increased risk of malignancy

-Hyper-IgM syndrome (XLR) Neutropenia Defective humoral immunity CD4OLG (Xq26)

-Schminke immuno-osseous dysplasia (AR) Neutropenia Spondyloeiphyseal dysplasia, SMARCAL1 (2q34-36) Pancytopenia nephrotic syndrome, Lymphopenia defective cellular immunity

-WHIM syndrome (AD) Neutropenia , hypogammaglobulinemia CXCR4 (2q21) Lymphopenia Infections, myelokathexis Neutrophil hyperplasia in the bone marrow, neutrophil nuclear hypersegmentation with thin filaments connecting pyknotic-appearing lobes

-Wiskott-Aldrich syndrome (XLR) Neutropenia Increased risk for acute WAS (Xp11.22-Xp11.3) Lymphopenia myeloid leukemia, diminished cellular immune function

*AD= autosomal dominant, AD dyskeratosis dyskeratosis congenita disorders are identified in italics; AR= autosomal recessive