Granulocyte function Flashcards
Describe the normal functions of neutrophils, including adherence, chemotaxis, ingestion, and degranulation/microbicidal activity.
Flow in laminar flow of blood
Interact with endothelial cell surface; interact w/ adhesion proteins; bring out of laminar flow onto surface to roll on endothelial cell. Tight adhesion, slips between endothelial surface, goes into sub endothelial cell matrix. Chemotaxis where neut is directed toward infection. Ingestion of the opsonized microbe (C3b or Ab). Fc receptors on neutrophil triggers ingestion, brings microbe in, fuses it with toxic granule
- Explain how neutrophil function is affected in the following disorders: leukocyte adhesion deficiency (LAD) I and II, actin dysfunction, specific granule deficiency, myeloperoxidase deficiency, Chediak-Higashi syndrome, and chronic granulomatous disease (CGD).
*LAD-1: Philia; Dec adherence to endothelial surface –> defect in movement to infected tissues
Gingivitis, periodontitis, umbilical cord sep
LAD-2: Philia; Dec rolling on endothelial surfaces as a prelude to tight adherence. RBC also affected, abnormal ABH antigens.
Actin: ↓ Chemotaxis, ↓ Ingestion, ↓ Spreading
*Chediak: Penia; Giant granules in all leukocytes. Abnormal/decreased degranulation and microbicidial activity. Major defect in movement
(Albinism, photophobia)
Specific: Decreased chemotaxis and microbicidal activity. Neutropenia (mild).
- Chronic: Neutrophilia. Defect in oxidase enzyme system. No toxic oxygen metabolites produced. Absent or reduced ability to kill coagulase positive bacteria and fungi (e.g., staph, E-coli). Recurrent purulent infection w/ catalase positive bacteria. Deadly!
- Myeloperoxidase: Partial or complete deficiency of MPO. Mild defect in killing bacteria, significant defect in killing CANDIDA. Inc fungal infection w/ diabetes
- Describe the NADPH oxidase enzyme system, techniques used to determine its activity, and the consequences of a defect in one of its components.
Initiates respiratory burst; Huge increase of oxidative metabolism and made into super oxide anion (O2-). In resting cell, components are disassembled. When cell is eating, complex assembled, NADPH provides electrons, e- are bound to O2 –> O2-, make ROS to kill microbe
DHR: Incubate cells with inactive fluorescent dye. Stimulate cell to make it think it’s eating. Start making Super Oxide, cell cleaves dye to make it fluorescent now. Measure on flow, peak moves to the right. Carrier will have 2 peaks
NBT: Test for CGD
- Drop of blood is suspended on a slide
- Add phagocytic stimulus (PMA), an activator of the oxidase
- Add NBT
- Neutrophils, take up NBT & PMA
- Normal result = dark purple (reduction of dye)
- CGD = no reduction, yellow
- Characterize the types of infections you might expect to see with defects of phagocyte function or complement.
Phagocytes
1) High rate of bacterial and fungal infections.
2) Infections with atypical or unusual microorganisms (e.g., Aspergillus, disseminated candida, lymphadenitis due to Serratia and other gram negative organisms, infections with Cepacia Burkholderi.
3) Catalase positive organisms in patients with CGD.
4) Infections of exceptional severity.
5) Peridontal disease in childhood.
6) Recurrent infections in areas of the body which interface with the microbial world.
Complement
1) Bacterial infections which might be seen with antibody deficiency (e.g., pyogenic organisms, H. influenzae, S. pneumoniae).
2) Terminal complement deficiencies (C5-C9) have problems with Neisseria organisms.
- Discuss tests which would characterize a phagocyte or complement problem. Differentiate between screening or confirmatory tests.
Phagocytes Screening • CBC, differential • Review of morphology • Bactericidal activity • Chemotaxis assay • Expression of CD11b/CD18 • NBT dye reduction or DHR oxidation.
Confirmatory/Detailed
• Adherence to inert surface or endothelial cells. Measurement of CD11b/CD18, L-selection, Sialyl LeX.
• Response to chemoattractants: shape change, change in direction, rate of movement. Actin assembly.
• Ingestion of labeled particles or bacteria. Degranulation of specific and azurophilic components.
• Bactericidal/candidicidal activity. Production of O2-, H2O2 other oxidants.
• Studies for specific molecular defects in oxidase or other cell constituents.
Complement
Screening
• C3, CH50
• Quantitative Ig’s, Lymphocyte numbers
Confirmatory/Detailed
• Measurement of specific complement components: alternative and classical pathways.
• Detailed evaluation of adaptive immune response.
- Discuss management strategies for patients with innate immune disorders.
1) Anticipation of infection and aggressive attempts to define the causative agent.
2) Surgical procedures for infected sites may be both diagnostic and therapeutic.
3) Prompt initiation of broad spectrum antibiotics covering a wide range of organisms, switching to specific coverage when microbial diagnosis is known.
4) For severe quantitative disorders of neutrophils, G-CSF may be used at a dose of 3 μg/kg/day to resolve the neutropenia (review from previous lecture).
5) Specific syndromes of neutrophil dysfunction may benefit from prophylactic antibiotics or cytokine therapy (e.g., INFγ for CGD).
6) Transplantation with hematopoietic stem cells has the capability to reconstitute neutrophil numbers and/or function.
7) Gene therapy: proof of concept studies has demonstrated reconstitution. Specific problems need to be resolved before a practical solution is achieved.
