Basics of Neutrophils And Disorders of Neutrophils

Basics of Neutrophils

• Basic functions of Neutrophils: Chemotaxis, Phagocytosis and Killing.

Chemotaxis

Process

1. Margination- Neutrophil coming close to the endothelial wall. Important substance: Fibrinogen. Positive acute phase reactant
2. Rolling- Weak interactions to endothelial wall. P selectin (Increased by histamine) and E selectin(Increased by IL1 and TNF) of endothelial site are important for rolling. Ligands for P selectin and E selectin: Sialyl Lewis x(Referred as CDI5S), called as Gylcamon CD34. On Neutrophilic side: L selectin bind with Gylcamon CD34. Sialyl lewis x maybe deficient in leukocyte adhesion deficiency type 2. P selectin is stored in Weibal Palade bodies of endothelium.
3. Adhesion- Strong interactions of endothelial wall. Cell adhesion molecules are needed. Referred to Integrin ligands-AKA ICAM-1 or VCAM-1, CD 54 and CD 106. All these are increased by IL1 and TNF. Receptors that bind with ICAM-1: LFA-1(Referred to 2 Integrin). LFA-1 has 2 components- CD18. Deficiency of CD18 will result in deficiency of LFA-1, results with Leukocyte adhesion deficiency type 1 genetic defect. CD11a receptors that bind with VCAM-1: VLA-4, α4/β1 integrin.
4. Diapedesis- Neutrophils tends to move in gaps. Important molecule: PECAM-1/CD-31, produces a collagenase called: Type 4 collagenase. Important for dissolution of endothelial base membrane.
5. Migration - Migrating in site of inflammation. This will contribute for acute inflammation.

Migration

• Need many other molecules- Chemo-attractants, LKT B4, Platelet activating factor, Chemokines- IL-8, KALLIKREIN, Complement fragments and bacterial products
• Defects of chemotaxis- LAD 1 due to mutation of gene called Integrin β2, molecule defective is CD18, primary adhesion defect  and diagnosed by flow cytometry. LAD 2 due to mutation of gene called FUCT2, molecule defective is Sialyl Lewis x and defective fucosylation of all selective ligands. Primary rolling defect and detected by genetic analysis. LAD 3 due to mutation of gene called FERMT3. All integrins are defective, activation defect, primary adhesion defect. Detected by genetic analysis.
• All are autosomal recessive- Recurrent infections, no pus formation, Leukocytosis, delayed umbilical cord separation- reason is you need neutrophils to remove the dead tissues and detach the umbilical cord. Here the neutrophil migration is defective. Keypoint for diagnosis- Glanzmann thrombasthenia seen in LAD-3.
• Lazy Leukocyte syndrome- Main problem is neutrophil chemotaxis, unknown cause, recurrent infection with neutropenia. Bone marrow granulocyte reserve is normal -Tested by Etiocholanolone test: Increased neutrophil mobilization.

Phagocytosis

Receptor ligand interactions.

Engulfment

• Due to production of pseudopodia form phagosomes- Cytoplasmic, Lysosomes fuse with phagosomes and form an area called phagolysosome. Killing typically happens in phagolysosome.

Killing

• Based on 2 basic processes:  Oxygen dependant and Oxygen Independent. Oxygen dependant involves production of ROS, referred to Respiratory Burst/Oxidative burst.

Respiratory Burst/Oxidative burst

• Production of oxygen free radical with help of NADPH oxidase enzyme. Whenever defective NADPH it’s going to result with Chronic Granulomatous disease. Conversion of oxygen to H2O2 by superoxide dismutase. After production of H2O2 addition of chloride will result in formation of  molecule called perchlorate(Hydroxy halide free radicals). Myeloperoxidase enzyme is needed. You can have myeloperoxidase deficiency. The pus which is blue green or purple green is due to this myeloperoxidase deficiency. These free radicals are generated to kill these organisms.
• But certain organisms which are catalase positive might convert the peroxide into water molecules by neutralizing them. Catalase enzymes are produced by Staphylococcus, Serratia, Nocardia, Aspergillus, Pasteurella, Burkholderia, E.Coli and Listeria. Mnemonic: SNAP BEL 
• Problems due to defective killing- Chronic Granulomatous disease- X linked defect (70%) most common, Autosomal recessive (30%), CYBB gene is mutated. Pathophysiology: NADPH oxidase deficiency. They cannot produce Respiratory Burst/Oxidative burst. They can phagocytose but can't kill the organism.
• Infections from catalase positive organisms, Recurrent bacterial and fungal infections from candida etc. The body will avoid infections by forming a wall from granuloma.

Diagnosing Chronic Granulomatous

• Normal neutrophil count: NBT test will be positive. Nitro blue tetrazolium chloride. A blood sample where the molecule added and if it contains NADPH oxidase, it will generate O2 free radical and it will change the color of NBT to blue color. If not present the color conversion won’t occur
• DHR 123: Dihydrorhodamine test: Currently preferred test, done by flow cytometry principle. A blood sample is taken and 2 components added (DHR and PMA stimulating(Phorbol myristate acetate)). DHR will be stimulated by the PMA. Once DHR taken by neutrophil, O2 free radical will produce a green fluorescence. Advantage is that it can detect the carrier state.

Treatment

• INF(Increase the O2 free radical). Allogenic stem cell Transplantation.

Also Read: Management of Inflammatory Bowel Disease (Ulcerative Colitis and Crohn's Disease) 

DISORDERS OF NEUTOPHILS

McLeod 

• Due to deletion of KELL antigen (XK): In certain cases, there might be deletion of Kell and CYBB gene too. Kell gene defect present in x chromosome, close proximity to the CYBB gene gives rise to Kell antigen.
• If deletion of gene is extended: Hemolytic anemia will contain numerous Acanthocytes. CNS (Seizure, Dementia etc), cardiac failure, peripheral nervous system will be affected, +/- Chronic Granulomatous disease(If CYBB is also deleted).

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Myeloperoxidase Deficiency

• Most common disorder of Neutrophil function. It is autosomal recessive. Acquired forms: Acute Myelogenous leukemia. 

MPO deficiency to become O2 free radical: NADPH, to become H2O2: Super-oxide Dismutase, to become hydrogen halide, you need Myeloperoxidase. Neutrophils will have normal oxidative burst and normal NBT and DHR 123 test. Patient will be typically asymptomatic, no neutrophilia and neutropenia. There will be only delayed microbicidal activity.

Chediak Higashi Syndrome 

• Due to LYST gene defect (Lysosomal trafficking regulator) aka Chediak Higashi Syndrome 1 gene, autosomal recessive inheritance, fuse the phagosome: For fusion you need the LYST gene. Usually a characteristic triad- Albinism, Recurrent Pyogenic infections, Peripheral Neuropathy. Fusion is important for release of granular contents.

Neutrophil Granules

• Neutrophil granules are modified lysosomes, O2 Independent killing. They are of three types- Granules/Azurophilic/Primary granules: Non specific because it can be seen in other granulocytes, lymphocytes and monocytes. These form very early in promyelocyte stage, contains: Acid hydrolase, BPI, Cathepsin G, Defensins, MPO and Peroxidase(Differentiates the Azurophilic granules from other lysosomes) 
• Secondary/ Specific granules: Form in myelocyte which are small and many in number while developed later contain:  Collagenase, Cathelicidin, NADPH Oxidase, Lactoferrin, ALP. Don’t stain with Romanowsky stain. Tertiary granules contain other cathepsins (Except G) and Gelatinases. Defective oxygen independent killing. Can have problems in defective platelet granule release, Bleeding diathesis. Few patients can enter accelerated phase with HLH like features, associated neutropenia, reduced chemotactic responses, reduced encasal activities. Only diagnosed by genetic test.

Treatment

• Allogenic stem cell transplantation and prophylactic antibiotics.

Diagnosis

• Giant azurophilic granules. This content cannot be released to phagosome. Can be seen in Melanocytes (Biopsy near hair or nerve).

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Benign 

• Pelger Heutomy, Reduced nuclear lobes. Reason: Autosomal Dominant due to laminin B receptor defects. Acquired conditions: Pseudo pelger heut, MDS, AML, CML, Myxedema, B12  & folate deficiency.

May–Hegglin anomaly (MHA)

Large basophilic cytoplasmic inclusions. Due to Autosomal Dominant- MYH 9 on chromosome 12. Associated giant platelets. Mild thrombocytopenia and mild bleeding.

Congenital Neutropenia 

• ELANE defects(ELA 2 defects)(AD). Types- severe congenital neutropenia, KOSTMAN syndrome, persistent high risk of increase of AML or MDS. Cyclical neutropenia happens every 21 days
• WHIM Syndrome- Warts, Hypogammaglobulinemi, Infections, Myelokathexis. Neutrophil pulled in bone marrow (Pooling of PMNS’s in BM). CXCRY for gain of function. Autosomal dominant disorder.

Also Read: Special Considerations in Diabetes Mellitus

Hope you found this blog helpful for your NEET SS Immunology Preparation. For more informative and interesting posts like these, keep reading PrepLadder’s blogs.