The Beloved Neutrophil: Its Function in Health and

Disease

Stem Cell Multipotent Progenitor

Myeloid Lymphoid

CMP IL-3, SCF, GM-CSF CLP MEP GMP

EPO TPO RBC Platelet Neutrophil Monocyte/ Basophil B-cells Macrophage Eosinophil T-Cells Mast cell NK cells

G-CSF M-CSF

GM-CSF, IL-3, SCF

IL-3 IL-5

Committed Progenitor

Mature Cell

SCF

Dendritic cells

PRODUCTION AND KINETICS OF NEUTROPHILS

CELLS % CELLS TIME

Bone Marrow: Mitotic Maturation/ Storage

Myeloblast Promyelocyte Myelocyte Metamyelocyte Band Seg

1 4

16

22 30 21

7 - 9 Days

3 – 7 Days

Vascular: Peripheral Blood Marginating Pool

Seg

2

3

6 – 12 hours

Tissue

Apoptosis and clearance by macrophages

? 0 – 3 days

PHAGOCYTOSIS

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

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

LFA-1 Mac-1 (CR3) p150,95 D2E2

D E

CD11a CD18

CD11b CD18

CD11c CD18

CD11d CD18

Cells All leukocytes

PMN, mono/mac, LGL

Dendritic

Mac, mono, PMN, T cell

Ligands ICAMs ICAM-1 C3bi Fibrinogen other

C3bi, other

ICAM-3, other

GRANULOCYTE CHEMOATTRACTANTS

Chemoattractants Source Activators Lipids PAF Neutrophils C5a, LPS, FMLP Endothelium LTB4 Neutrophils FMLP, C5a, LPS Chemokines (D) IL-8 Monocytes, endothelium LPS, IL-1, TNF, IL-3 other cells Gro D, E, J Monocytes, endothelium IL-1, TNF other cells NAP-2 Activated platelets Platelet activation Others FMLP Bacteria C5a Activation of complement

Other Important Receptors on PMNs

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

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 elastase) Lysozyme Digestion of bacterial cell wall Defensins and bactericidal/proteins 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)

x Frequent and/or unusually severe bacterial, fungal infections

- Skin, lymph nodes, lungs (portals of entry); other sites via bloodstream or tissue extension

x Unusual site - e.g. liver or brain abscess

x Recurrent/chronic gingivitis, aphthous ulcers

x 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: Leukocyte Adhesion Deficiencies (LAD) I, II, III

Chemotaxis: LADI HyperIgE Syndrome Chediak-Higashi Actin defects

Phagocytosis: LADI Chediak-Higashi

Killing: Chronic granulomatous disease Specific granule deficiency Chediak-Higashi MPO deficiency (with DM)

LEUKOCYTE ADHESION DEFICIENCY In Vitro Leukocyte Functional Abnormalities

Clinical Features

x Pediatric age group x Delayed umbilical cord separation x Persistent leukocytosis x Defective neutrophil mobilization

(reduced pus formation) x Impaired wound healing x Recurrent (life-threatening) bacterial

and sometimes viral infections x PMN/Mo adherence and spreading x PMN aggregation

Lekstrom-Himes JA, Gallin JI. Immunodeficiency 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 mutation in CD18 subunit of E2 integrin, leading to complete or partial loss of E2 integrin expression Pathogenesis Defects in adhesion, migration, phagocytosis Clinical Omphalitis, periodontitis, skin/soft tissue infections, pneumonia, sepsis, poor wound healing, delayed cord separation. Staph, gram negatives Labs Neutrophilia, absent CD11/CD18 (including Mac-1) Management HSCT, otherwise supportive care

SUMMARY OF CGD Incidence: 4 per million births Infections: x Pneumonia (70%) Aspergillus x Supportive Adenitis (53%)

Staphylococcus x Subcutaneous Abscess (42%)

Staphylococcus x Liver Abscesses (27%) Staphylococcus x Osteomyelitis (25%) Serratia x Sepsis (18%) Salmonella

INFECTIONS IN CGD Pathogen Presentations

Bacterial � Staphylococcus aureus

Soft tissue infection, 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 � Candida species

Pneumonia, Osteomyelitis, Brain abscess Sepsis, Soft tissue infection, Liver abscess

Lung resection for persistent Aspergillus

Pathology: Chronic inflammatory infiltrate, foci of neutrophils

Silver stain: Aspergillus hyphae

Examples of Infectious Complications of CGD

Impetigo

Lymphadenitis Aspergillus

pneumonia

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

SUMMARY OF CGD

x Other complications: Gastric Outlet Obstruction (15%)

Urinary Tract Obstruction (10%) Colitis (17%) x 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 inflammation, including GI tract Labs Absent or markedly reduced NADPH oxidase activity Management Prophylactic trimethoprim/sulfa, itraconazole, IFNJ; Aggressive/prolonged treatment of infections; Prednisone/ immunisuppr for inflammation

Neutropenia

• 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 bone marrow?

• Production vs. destruction.

Epidemiology

• Acute neutropenia occurs frequently.

• Congenital neutropenia: ~ 2 per million.

• Cyclic neutropenia: ~ 0.6 per million.

Suspicion of Neutropenia

• Acute severe bacterial infections.

• History of recurrent infections.

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

• Pneumonia, peritonitis, 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.

Antigens

Previous

Names Carrier Glycoproteins

Allele Frequency

(%) Asians*

Allele Frequency

(%) Africans+

Allele Frequency

(%) Whites

Clinical Significance

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

Human Neutrophil Alloantigens

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) mutations

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 neutrophil

elastase

• ELANE (ELA2) mutations found in essentially 90% of cyclic neutropenia

• NOT associated with an increased risk of AML

0

1000

2000

3000

4000

5000

6000

0 21 42 63 84 105 126

Congenital Neutropenia

0

1000

2000

3000

4000

5000

6000

0 21 42 63 84 105 126

ANC AMC

Cyclic Neutropenia

AMC

Peri

ph

eral

Leu

kocy

te C

ou

nt

(co

un

ts/µ

l)

Dynamics of the Absolute Monocyte Count

Days ANC

Table 3: Classification of Congenital Neutropenia Disorders

Main hematologic Other clinical Gene 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 telomere dysfunction -Shwachman-Diamond (AR) Neutropenia Exocrine SBDS (7q11.22) Aplastic anemia pancreas deficiency MDS/AML Metaphyseal dysostosis -Dyskeratosis congenita (XLR) Pancytopenia Abnormal skin pigmentation, DKC1(Xq28) -Dyskeratosis congenita (AD) MDS/AML nail dystrophy, TERC (3q26)* -Dyskeratosis congenital (AR) oral leukoplakia, TERT (5p33)* epiphora, pulmonary fibrosis, TINF2 (14q11.2) short stature, hair loss, NOP10 (15q14-q15) developmental delay, NHP2 (5q35.3) squamous cell carcinoma of TCAB1(17q13.1) head and neck

Disorders of Metabolism -Reticular dysgenesis (AR) Neutropenia Sensorineural hearing loss AK2 (1p31-p34) Lymphopenia -Barth syndrome (XLR) Neutropenia Cardiomyopathy TAZ1 (Xq28) -Glycogen storage disease type 1b (AR) Neutropenia Hypoglycemia, G6PT1 (11q23) hyperlipidemia, hyperuricemia, growth retardation, osteopenia, renal hypertrophy -Glucose-6-phosphatase Neutropenia Cardiac abnormality, G6PC3 (17q21) catalytic subunit 3 (AR) Thrombocytopenia prominent superficial venous pattern, hepatosplenomegaly, cryptorchidism, microcephaly -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 RAB 27A (15q14.1) and hair -Hermansky-Pudlak type II (AR) Neutropenia Oculocutaneous albinism AP3B1 (5q14.1)

-p14 deficiency (AR) Neutropenia, Short stature, MAPBPIP (1q21) Decreased B&T hypopigmentation 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 Warts, 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, Monocytopenia diminished cellular immune function

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