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Luis Lopez, a 16 year old high school student with chronic bacterial and fungal infections |
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Describe the defect in CGD and how it leads to recurrent
bacterial and
Luis Lopez, a 16 year old high school student had a summer job working with a landscaping company. His job involved spreading mulch in garden beds. At the end of August of that summer he developed shortness of breath, a persistent cough and chest pain. He was admitted to the hospital because of impending respiratory failure. A chest X-ray revealed the presence a large opacity in the right lung (FIGURE). One lesion was aspirated with a fine needle and the aspirate was stained and observed to contain aspergillus hyphae. When this was cultured it was found to contain Aspergillus fumigatus.
Luis was given intravenous amphotericin B and started on a mechanical ventilator. He improved slowly over 3 months, however, during his stay at the hospital he contracted two additional respiratory infections with Pseudomonas aeruginosa and Streptococcus faecalis. These infections were treated with appropriate antibiotics.
| Blood cell count | Serum Immunoglobulins |
Chest X-Ray |
| 12,000 ul (normal 5000-10,000 ul) | serum IgG level was 1725 mg dL (upper limit of normal is 1500 mg dL) |  |
| 60% neutrophils | IgM and
IgA were in the high normal range at 250 and 175 mg dL
respectively |
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| 30% lymphocytes | ||
| 5% monocytes |
When he was first admitted, Luis’s white blood cell count was 12,000 ul-1 (normal 5000-10,000 ul-1). He had 60% neutrophils, 30% lymphocytes, and 5% monocytes, all of which were in the normal range. Luis’s mother informed the attending physician that when he was 13, Luis developed granulomas on his skin following a Staphlyococcus aureus infection.
Because of his infections, which were unusual for a 16 year old adolescent, his serum immunoglobulins were measured. The serum IgG level was 1725 mg dl-1 (upper limit of normal is 1500 mg dl-1). IgM and IgA were in the high normal range at 250 and 175 mg dl-1 respectively. Since no defect could be found in his humoral immunity, his white cell function was tested with a nitro blue tetrazolium (NBT) slide test. His white blood cells failed to reduce NBT.
Luis has three older sisters and two brothers. They all had an NBT test performed on their blood. One brother Carlos, 7 years old, also failed to reduce NBT. His mother reported that he had had a perirectal abscess in infancy but had otherwise been well. Half of the neutrophils from Luis’s mother and one sister were able to reduce NBT and the other half were not.
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NBT reduction was normal for Luis’s other two sisters and his other brother.
Further studies with Luis’s and Carlos’s neutrophils revealed that the rate of hydrogen peroxide production was 0.17 and 0.030 nmol/min-1 10-6 cells respectively (normal 6.35 nmol/min-1 10-6 cells ). The cytochrome b content in both brothers’ granulocytes was < 1.0pmol/mg-1 protein (normal control 101/pmol mg-1 protein). Both Luis and Carlos were started on treatment with injections of interferon-g.
Internet Links
Nitroblue tetrazolium test
QUESTIONS:
1. What type of immunodeficiency disease does Luis have? What is the genetic defect in this disease and what are the consequences of this defect? Is Luis’s disease X-linked?
ANSWER 1.
Luis suffers from Chronic Granulomatous Disease (CGD). The genetic defect in this disease is a mutation in NADPH oxidase. This defect results in an inability of phagocytic cells to undergo a respiratory burst which leads to the production of H2O2. When phagocytes ingest microorganisms they become activated and their microbicidal functions are induced. One of these microbicidal activities involves the production of hydrogen peroxide (H2O2) and superoxide radicals which raise the pH of the phagosomes facilitating the release of phagosomal enzymes that attack the ingested microbes.
NADPH oxidase catalyzes the following reaction:
NADPH + 2O2 NADP+ + 2O2+ H+
The superoxide then dismutates to produce hydrogen peroxide as follows:
2H+ + 2O2 H2O2 + O2
NADPH oxidase is a large, multisubunit enzyme complex located in the membrane of the phagosome. It is assembled from components in the membrane and cytosol, in response to a phagocytic stimulus. The two membrane components form cytochrome b558. Two chains comprise cytochrome b558: a heavy chain, gp91phox, and a light chain, p21phox. The gp91phox gene is encoded on chromosome X. The cytosol components are encoded by several genes that map to autosomal chromosomes. Four different genetic defects affecting various components of NADPH oxidase have been identified and they all result in CGD. The most common form of the disease is the X-linked. It is likely that Luis’s disease is X-linked since his brother Carlos also has the disease and since his mother and one sister appear to be carriers according to the NBT test.
Patient’s with CGD are highly susceptible to low virulence bacteria and fungi such as
Staphylococcus aureus, Pseudomonas aeruginosa, and
Aspergillus fumigatus, due to defective microbicidal mechanisms of their phagocytes. Without a functional NADPH oxidase they cannot produce hydrogen peroxide or superoxide radicals that are toxic to intracellular microorganisms. Inability to clear the opsonized microorganisms often results in granuloma formation.
2. What is the basis for the NBT test (discuss how it works)? Why are half of the neutrophils from Luis’s mother and sister able to reduce NBT and the other half are not?
ANSWER 2.
A diagnosis of CGD can be made from the
nitro blue tetrazolium test (NBT). The NBT dye is initially pale yellow and transparent but when it is reduced, it becomes insoluble and deep purple in color. To test for CGD, a drop of a patient’s blood is suspended on a slide and a phagocytic stimulus such as phorbol myristate acetate (PMA) is added. Next a drop of NBT is added and neutrophils and other phagocytic cells will take up the dye and the PMA. If NADPH oxidase is present in the phagocytes of the blood sample then it will catalyze the release of hydrogen from NADPH and this will reduce the NBT to insoluble formazan which is purple. The blue stained neutrophils can easily be seen microscopically. In CGD, no reduction of NBT is observed and the neutrophils fail to stain purple.
Luis’s mother and sister are heterozygous carriers for the X-linked CGD defect. That implies that they have one normal X chromosome and one with the defective gene on it. Due to X inactivation, the defective X chromosome is randomly inactivated in approximately 50% of the carrier’s phagocytic cells while the normal X is inactivated in the other 50%. Therefore half of Luis’s mother’s and sister’s neutrophils stain with reduced NBT and the other half do not.
3. How and under what conditions do granulomas form? What is the cellular composition of a granuloma?
ANSWER 3.
Sustained exposure of phagocytes to pathogens or inability of phagocytes to clear intracellular pathogens can lead to a chronic local inflammation called a
granuloma. Granulomas consist of a core of infected macrophages, many of which are fused to form multinucleated giant cells, surrounded by large macrophages called epithelioid cells. These can in turn be surrounded by CD4 TH1 cells which help activate the macrophages.
Granulomas can frequently form in CGD because without NADPH oxidase, macrophages are unable to destroy ingested pathogens. This chronic exposure to a pathogen that they cannot kill, forces the macrophages to wall off the pathogen in an effort to contain it.
4. How do you explain the elevated immunoglobulin levels in Luis’s blood?
ANSWER 4.
Because Luis is persistently exposed to pathogens he cannot eliminate, he makes more immunoglobulins than a normal person. All chronic infections result in
hypergammaglobulinemia.
5.
Children with CGD are susceptible to many bacterial and fungal infections but they do not seem to develop pneumococcal infections? Can you explain the possible reason for this?ANSWER 5.
In CGD, macrophages can phagocytize microorganisms but they cannot kill catalase positive microorganisms such as
Staphylococcus and Salmonella. Therefore patients with CGD frequently succumb to these pathogens. This is because the presence of the
catalase enzyme (catalase converts H2O2 into water and oxygen) from the bacteria and the absence of NADPH oxidase from the host macrophages, prevents the production of H2O2 and superoxide radicals which are toxic to intracellular bacteria. In the absence of these microbicidal products the intracellular catalase positive microorganisms can persist in the phagocytes and cause chronic inflammation in the host. In contrast, catalase negative microorganisms such as Streptococcus (ie. Streptococcus pneumoniae) cannot break down H2O2 and so it accumulates in the macrophages and neutrophils that phagocytose these microorganisms. In CGD patients, catalase negative microorganisms cannot be destroyed by H2O2 from the phagocytes since there is none produced, but they can be destroyed by H2O2 that is endogenously produced by the bacteria.
6.
How does CGD differ from other phagocytic immunodeficiency diseases (list the other diseases and describe their defects)? What other types of immunodeficiency diseases can result in recurrent bacterial infections?
ANSWER 6.
Deficiencies in the enzymes glucose-6-phosphate dehydrogenase (G6PD) and myeloperoxidase also result in impaired production of toxic oxygen metabolites and therefore impaired respiratory burst and impaired bacterial cell killing by macrophages and
neutrophils.
Chediak Higashi Syndrome (CHS)
is an autosomal recessive phagocyte immunodeficiency disease that results from defective fusion of endosomes and lysosomes leading to impaired intracellular killing. Recurrent bacterial infections and granulomas are common in this disease.
Leukocyte adhesion deficiency diseases
(LAD I and
LAD II) are caused by the lack of certain functional adhesion molecules on phagocytes. This prevents the phagocytes from migrating to where they are needed and results in impaired wound healing. LAD I immunodeficiency arises from mutations in the gene that encodes CD18 which is the b chain of the integrins LFA-1 and the complement receptors, CR2 and CR4. A defect affecting LFA-1 results in reduced transendothelial migration. Defects in CR2 and CR4 also result in impaired opsonization of complement coated bacteria. LAD II immunodeficiency causes abnormal fucose metabolism which leads to reduced expression of fucosylated ligands on the surface of phagocytes. This prevents the interaction of these cells with selectins on endothelial cell walls and impairs macrophage and neutrophil migration along the endothelial surface. Patients with LAD deficiencies also get frequent bacterial infections.
Although phagocytosis by macrophages and neutrophils is the main way that the immune system destroys bacteria, antibodies and complement also facilitate the removal of bacteria. B cell immunodeficiency diseases and SCID immunodeficiency diseases lead to recurrent bacterial infections.
