1. Phagocytosis and diseases of phagocytic cells
Kammi Henriksen, MD
Department of Pathology

2. Outline Clinical case Overview of leukocyte activation
Sequence of events in phagocytosis
Diseases of phagocyte function

3. Clinical case
A 67 year old man with long-standing and poorly controlled type 2 diabetes mellitus presents to his primary care physician complaining of a foul-smelling open wound on the bottom of his foot
Physical examination reveals a large ulceration involving the skin and soft tissue overlying the first and second distal metatarsals
Associated with purulent discharge, redness, warmth, and swelling
Probing reveals the presence of exposed bone in the depth of the ulcer, suspicious for osteomyelitis
Lower limb pulses are moderately diminished
Touch sensation is virtually absent

4. Lab tests and Imaging
Lab testing is significant for leukocytosis, elevated ESR and CRP, and elevated hemoglobin A1c
X-ray findings consistent with osteomyelitis
Marked bone lysis of the distal first metatarsal, first proximal phalanx, and first distal phalanx
Marked soft tissue swelling along the medial foot and surrounding the first toe joints
The patient subsequently undergoes ray amputation

5. Pathology
Sections from the edge of the skin wound show ulceration of the squamous epithelium with dermal fibrosis and acute and chronic inflammatory cell infiltrate

6. Pathology
Sections from the first metatarsal head show that the bone marrow has been replaced by acute and chronic inflammatory cells including abundant neutrophils
Also noted are fragments of necrotic bone

7. Diabetic foot infection
Most important chronic complication of DM
One of the most common causes of hospitalization
Often results in amputation, osteomyelitis, and death
Moderate or “non-limb threatening”
Superficial, cellulitis <2.0 cm in largest dimension, without evidence of serious ischemia, systemic toxicity, or bone and/or articular involvement
Serious or “limb-threatening”
Deep ulceration, cellulitis >2.0 cm, evidence of serious ischemic, systemic toxicity, or bone/joint involvement
Monomicrobial or polymicrobial
Staph, Strep, Enterococci, Enterobacteria, anaerobic bacteria

8. Osteomyelitis in the diabetic foot
Osteomyelitis (OM) is a common complication of diabetic foot ulcers and/or diabetic foot infection
Underlying OM is seen in 20% of patients with diabetic foot infection
Frequently missed/underdiagnosed
Risk of developing OM increases with ulcers >2cm or foot ulcer with exposed bone or joints
The risk for amputation in acute diabetic infections is four time higher with OM that soft tissue infection alone
Presence of OM requires longer duration of antibiotic therapy and longer duration of hospital stay

9. Infections in patients with diabetes mellitus
Diabetics are prone to infectious complications
Account for up to 22% of deaths in one series
DM has been associated with reduced response of T cells, neutrophil function, and disorders of humoral immunity
Hyperglycemia  increased infection risk and neutrophil dysfunction
May relate to aldose reductase pathway (polyol pathway)
Excess glucose is converted to sorbitol, with consumption of NADPH
Insufficient reducing equivalents (NADPH) may lead to defective oxidative burst in insulin-dependent cells, including neutrophils

10. Pathophysiology of infections associated with DM
Deficiency of C4
Monocytes secrete less IL-1 and IL-6 in response to LPS
Decreased mobilization of PMNs, chemotaxis, and phagocytic activity in hyperglycemia
Glycosylation of Igs, which may harm functions
Main pathogenetic mechanisms:
Hyperglycemic environment increases the virulence of some pathogens
Lower production of interleukins in response to infection
Reduced chemotaxis and phagocytic activity
Immobilization of neutrophils
Glycosuria
GI and urinary dysmotility

11. Overview of leukocyte activation

12. Leukocyte activation
Recognition of microbes or dead cells induces several responses known as leukocyte activation
Signaling pathways are triggered, resulting in increased cytosolic Ca2+ and activation of enzymes
Functional responses most important for destruction of microbes are phagocytosis and intracellular killing

13. Phagocytosis and endocytosis
Endocytosis: portions of the cell membrane invaginate -> membrane-bound cytoplasmic vesicles
Phagocytosis: a specialized version of endocytosis involving the internalization of solids such as bacteria
Pinocytosis (“cell drinking”): ingestion of fluids and solutes via small vesicles (<150 nm)
Exocytosis: the reverse of endocytosis
Phagocytosis

14. Phagocytosis Performed by specialized “professional” cells
Neutrophil
Monocyte/macrophage
3 sequential steps:
Recognition and attachment of the particle to be ingested by the leukocyte
Engulfment and formation of a phagocytic vacuole
Killing or degradation of the ingested material

15. Phagocytosis in Brief

16. Opsonization
Molecular mechanism whereby molecules, microbes, or dead cells are chemically modified to have stronger interactions with cell surface receptors on phagocytes
With the antigen coated in opsonins, binding to immune cells is greatly enhanced
Major opsonins: IgG antibodies, C3b breakdown component of complement, mannose-binding lectin
Opsonization also mediates phagocytosis via signal cascades from cell surface receptors

17. Overview of phagocytosis
Attachment and binding of Fc and C3b to receptors on the leukocyte membrane
Engulfment
Fusion of lysosomes with phagocytic vacuoles
Destruction of ingested particles within the phagolysosomes.
Note that during phagocytosis, granule contents may be released into extracellular tissues.

18. Phagocytosis and intracellular destruction of microbes

19. Phagocytic receptors Mannose receptors Scavenger receptors
Bind terminal mannose and fucose residues on microbial cell walls (sugars which are not on host cells)
Scavenger receptors
Bind a variety of microbes in addition to modified low-density lipoprotein (LDL) particles
Macrophage integrins
Mac-1 (CD11b/CD18)
Receptors for various opsonins
Complement receptors, Fc receptors

20. Phagocytic receptors

21. Phagocytosis and intracellular destruction of microbes

22. Engulfment Particle binds phagocyte receptor
Extensions of cytoplasm (pseudopods) flow around it
Plasma membrane pinches off to form a vesicle (phagosome) enclosing the particle
Phagosome fuses with a lysosomal granule
Discharge of granule contents into phagolysosome
Phagocyte may also release granule contents into extracellular space

23. Phagocytosis and intracellular destruction of microbes

24. Intracellular destruction of microbes and debris
Killing mechanisms are normally sequestered in lysosomes, thus segregated from cell’s cytoplasm and nucleus
Oxygen-dependent killing:
Reactive oxygen species
Reactive nitrogen species (derived from nitric oxide)
Oxygen-independent killing:
Lysosomal enzymes
Other granule enzymes

25. Reactive oxygen species (ROS)
ROS are produced by the rapid assembly and activation of a multicomponent oxidase: phagocyte NADPH oxidase
In response to phagocyte activation, the protein components assemble on the phagolysosome membrane, so ROS are produced within
“Respiratory burst” – NADPH is oxidized and oxygen is reduced to superoxide anion
From there, additional ROS are generated (see below)
Oxygen-derived free radicals bind to and modify cellular lipids, proteins, and nucleic acids -> destroy microbes
Phagocytes (i.e., neutrophils and macrophages) require an enzyme to produce reactive oxygen species to destroy bacteria after they are ingested (phagocytosis), a process known as the respiratory burst. This enzyme is termed "phagocyte NADPH oxidase" (PHOX). This enzyme oxidizes NADPH and reduces molecular oxygen to produce superoxide anions, a reactive oxygen species. Superoxide is then disproportionated into peroxide and molecular oxygen by superoxide dismutase. Finally, peroxide is used by myeloperoxidase to oxidize chloride ions into hypochlorite (the active component of bleach), which is toxic to bacteria. Thus, NADPH oxidase is critical for phagocyte killing of bacteria through reactive oxygen species.
(Two other mechanisms are used by phagocytes to kill bacteria: nitric oxide and proteases, but the loss of ROS-mediated killing alone is sufficient to cause chronic granulomatous disease.)
Defects in one of the four essential subunits of phagocyte NADPH oxidase (PHOX) can all cause CGD of varying severity, dependent on the defect. There are over 410 known possible defects in the PHOX enzyme complex that can lead to chronic granulomatous disease.[3]
Notably, the neutrophilic enzyme myeloperoxidase
(MPO) plays a role in this process

26. Nitric oxide (NO)
Inducible nitric oxide synthase (iNOS) is induced when phagocytes are activated by cytokines or microbial products
Reacts to form nitrogen-derived free radicals which also attack and damage lipids, proteins, and nucleic acids
Reactive oxygen and nitrogen species have overlapping actions
NO also relaxes vascular smooth muscle and promotes vasodilation

27. Oxygen-Independent Killing
Lysozyme
Cleaves the (1-4) glycosidic linkage between N- acetylglucosamine and N acetylmuramic acid
Role in bacterial killing: mainly synergistic
Cathepsin G
Protease that digests bacterial proteins
Also microbicidal when denatured
Bactericidal Permeability Increasing Protein (BPIP)
Cationic (pK ≈ 9.6); Specific for g-negative bacteria
Inserts into outer membrane of g-negatives, increases membrane permeability
Also acts synergistically with oxygen radicals, etc
Major basic protein (MBP) of eosinophils
Not very bactericidal but quite toxic to many helminths and other parasites
Defensins
Small (MW<4000), cationic, cystine rich peptides found in macrophages and neutrophils
Weakly bactericidal
Lactoferrin: iron-binding glycoprotein for the production of hydroxyl radical

28. Neutrophil extracellular traps (NETs)
Healthy neutrophils with red
nuclei and green cytoplasm
EM of bacteria (Staph)
trapped in NETs
Release of nuclear material,
forming NETs (and loss of nuclei)
Produced by neutrophils in response to pathogens , inflammatory mediators, complement, and ROS
Extracellular fibrillar networks with a high concentration of antimicrobial substances at site of infection
Prevent spread of microbes by physically trapping them
Consist of a viscous meshwork of nuclear chromatin (might be a source of nuclear Ags in systemic autoimmune diseases – SLE)
In the process of NET formation, nuclei are lost and neutrophils die
NETs detected in the blood in patients with sepsis

29. Leukocyte-mediated tissue injury
Leukocytes are important causes of injury to normal cells and tissues under several circumstances:
Part of normal defense reaction against infectious microbes, when adjacent tissues suffer collateral damage (e.g. prolonged host response in certain infections such as TB)
Autoimmune diseases – inflammation inappropriately directed against host tissues
Allergies – host reacts excessively to usually harmless environmental substances

30. Leukocyte-mediated tissue injury
The mechanisms by which leukocytes damage normal tissues are the same as the mechanisms involved in antimicrobial defense
Once leukocytes are activated, effector mechanisms do not distinguish between offender and host
ROS, NO, and lysosomal enzymes produced within the phagolysosome are also released into the extracellular space
Capable of damaging normal cells and endothelium
Antioxidant mechanisms in serum, tissue fluids, and host cells protect against some of this damage
Lysosomal enzymes are normally controlled by a system of antiproteases in the serum and tissue fluids
a1-antitrypsin - major inhibitor of neutrophil elastase

31. Diseases of phagocyte function
Leukocyte adhesion deficiencies (LAD)
Chronic granulomatous disease (CGD)
Chediak-Higashi syndrome
Myeloperoxidase deficiency
Altered neutrophil and monocyte function in other medical illnesses

32. Leukocyte Adhesion Deficiencies (LAD)
First recognized as a clinical entity in the 1970s
Classic description: bacterial infections, defects in neutrophil adhesion with inability to form pus, delayed umbilical cord sloughing
Rare autosomal recessive disorder characterized by recurrent infections
LAD1: deficiency of b2 integrin CD11/CD18
Ligand for ICAM-1 on endothelium (required for stable adhesion)
Impaired neutrophil adhesion and chemotaxis
LAD2: deficiency of neutrophil sialyl Lewis X-modified glycoprotein
Inability of neutrophils to adhere to E-selectin on endothelial cells
Defective neutrophil rolling and adhesion

34. Leukocyte Adhesion Deficiencies (LAD)
Clinical manifestations
Recurrent skin and mucous membrane infections from Staph aureus, Pseudomonas, other Gram negatives, Candida
Skin lesions are often gangrenous because lack of pus formation
Labs
CBC show extremely elevated levels of neutrophils because they are unable to leave blood vessels
Flow cytometry for absent/reduced CD18 or SLeX expression

35. Flow cytometry in LAD1
Reduced expression of integrin (CD18) on CD3+ T cells in a patient with LAD1 (right)
as compared to normal control (left).

36. Subtypes of LAD

37. Chediak-Higashi Syndrome (CHS)
Rare autosomal recessive disorder, mutation in CHS1 gene
Mutation of a lysosomal trafficking regulator protein, leading to defective fusion of phagosomes and lysosomes
Neutropenia, defective degranulation, and delayed microbial killing
Clinical manifestations:
Incomplete oculocutaneous albinism: light skin, silvery hair, loss of pigment in iris and retina, photophobia, nystagmus, increased red reflex
Recurrent pyogenic infections of skin, respiratory tract, and mucous membranes (recurrent gingivitis and peridontitis)
Neuropathy
Infections in CHS patients are very serious; few patients live to adulthood

38. Chediak-Higashi Syndrome (CHS)
Many children with CHS reach a stage known as the accelerated phase (lymphoma-like syndrome)
Defective WBCs divide uncontrollably and invade organs, resulting in hepatomegaly and pancytopenia
Fever, recurrent severe infections, abnormal bleeding, and organ failure
Usually life-threatening in childhood
CHS is characterized by large lysosome vesicles in phagocytes (neutrophils, monocytes, BM myeloid cells)
Disordered intracellular trafficking -> impaired lysosome degranulation with phagosomes
Melanocytes contain giant melanosomes that fail to disperse melanin

39. Chediak-Higashi Syndrome (CHS)
Left: a boy with Chédiak-Higachi syndrome and partial albinism
Right: hair from a patient with Chédiak-Higachi syndrome (the black spots are giant melanosomes)

40. Chediak-Higashi Syndrome (CHS)
Silvery light hair (left) and giant granules in neutrophils (right)

41. Chediak-Higashi Syndrome (CHS)
Giant granules in monocytes and neutrophil of a patient with CHS

42. Chronic granulomatous disease (CGD)
Diverse group of hereditary diseases with defective respiratory burst in neutrophils
First described in 1950 in 4 boys as “fatal granulomatosus of childhood”
Affects 1 in 200,000 in the US (20 new cases per year)
Molecular defects are in the genes encoding the NADPH oxidase
X-linked (2/3rd of cases) and autosomal recessive forms
Affected individuals can ingest but not kill organisms
Name derives from macrophage-rich chronic inflammatory reaction that tries to control the infection when the initial neutrophil defense is inadequate
Collections of activated macrophages wall off the microbes, forming granulomas

43. Chronic granulomatous disease (CGD)
Clinical manifestations
Recurrent pneumonia, skin infections, hepatic abscesses, osteomyelitis
Typically catalase positive organisms such as S. aureus
Leads to formation of granulomata in many organs
Eventually patients develop sequelae of chronic infections
Anemia of chronic disease, hepatosplenomegaly, gingivitis, GI narrowing, restrictive lung disease
Diagnosis: nitroblue tetrazolium test
Prognosis better now with prophylactic Abx; average patient survives at least 40 years
Phagocytes (i.e., neutrophils and macrophages) require an enzyme to produce reactive oxygen species to destroy bacteria after they are ingested (phagocytosis), a process known as the respiratory burst. This enzyme is termed "phagocyte NADPH oxidase" (PHOX). This enzyme oxidizes NADPH and reduces molecular oxygen to produce superoxide anions, a reactive oxygen species. Superoxide is then disproportionated into peroxide and molecular oxygen by superoxide dismutase. Finally, peroxide is used by myeloperoxidase to oxidize chloride ions into hypochlorite (the active component of bleach), which is toxic to bacteria. Thus, NADPH oxidase is critical for phagocyte killing of bacteria through reactive oxygen species.
(Two other mechanisms are used by phagocytes to kill bacteria: nitric oxide and proteases, but the loss of ROS-mediated killing alone is sufficient to cause chronic granulomatous disease.)
Defects in one of the four essential subunits of phagocyte NADPH oxidase (PHOX) can all cause CGD of varying severity, dependent on the defect. There are over 410 known possible defects in the PHOX enzyme complex that can lead to chronic granulomatous disease.[3]

44. Chronic granulomatous disease (CGD)
Simple, rapid diagnostic test for diseases of phagocyte function
Test depends on direct reduction of NBT to the insoluble blue compound
formazan by NADPH oxidase
The higher the blue score, the better the cell is at producing ROS
Phagocyte NADPH oxidase defect -> unable to make ROS or radicals
NBT test is negative in CGD (does not turn blue)

45. Chronic granulomatous disease (CGD)
Granuloma-like lesion caused by a normally pyogenic bacterium
in a patient with Chronic Granulomatous Disease.

46. Myeloperoxidase (MPO) deficiency
One of the more common immunodeficiencies (1:4,000)
AR disorder featuring deficiency (either in quantity or function) of MPO
MPO is usually present in azurophilic granules of neutrophils -> catalyses conversion of hydrogen peroxide to hypohalous acid -> amplifies toxicity of the ROS generated during the respiratory burst
In vitro, MPO deficient cells take twice as long to kill pathogens
Normal NBT test because they still have NADPH oxidase activity
Classically presents with immune deficiency (especially Candida infections)
Majority of individuals show no signs of immunodeficiency
Suggests the role of MPO in the immune response must be redundant to other mechanisms of intracellular killing of phagocytosed bacteria

47. Defects in leukocyte function
Disease
Defect
Defects in Leukocyte Function
Leukocyte adhesion deficiency 1
Defective leukocyte adhesion because of mutations in β chain of CD11/CD18 integrins
Leukocyte adhesion deficiency 2
Defective leukocyte adhesion because of mutations in fucosyl transferase required for synthesis of sialylated oligosaccharide (receptor for selectins)
Chédiak-Higashi syndrome
Decreased leukocyte functions because of mutations affecting protein involved in lysosomal membrane traffic
Chronic granulomatous disease
Decreased oxidative burst
 X-linked
Phagocyte oxidase (membrane component)
 Autosomal recessive
Phagocyte oxidase (cytoplasmic components)
Myeloperoxidase deficiency
Decreased microbial killing because of defective MPO-H2O2system

48. Altered phagocyte function in other medical diseases

49. Summary: Leukocyte activation and removal of offending agents
Leukocytes can eliminate microbes and dead cells by phagocytosis, followed by their destruction in phagolysosomes
Destruction is caused by free radicals (ROS, NO) generated in activated leukocytes and lysosomal enzymes
Neutrophils can extrude their nuclear contents to form extracellular nets that trap and destroy microbes
Enzymes and ROS may be released into the extracellular environment
The physiologic mechanisms that function to eliminate microbes and dead cells are also capable of damaging normal tissues (pathologic consequences)
Defects of leukocyte function can cause diseases characterized by recurrent infections