Scandinavian Journal of Infectious Diseases

ISSN: 0036-5548 (Print) 1651-1980 (Online) Journal homepage: http://www.tandfonline.com/loi/infd19

Recurrent Pseudomonas Infection Associated with Neutrophil Dysfunction Ann O. Shigeoka & Harry R. Hill To cite this article: Ann O. Shigeoka & Harry R. Hill (1978) Recurrent Pseudomonas Infection Associated with Neutrophil Dysfunction, Scandinavian Journal of Infectious Diseases, 10:4, 307-311, DOI: 10.3109/inf.1978.10.issue-4.09 To link to this article: http://dx.doi.org/10.3109/inf.1978.10.issue-4.09

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Date: 20 April 2016, At: 04:40

Scand J Infect Dis 10: 307-3 1 1, 1978

Case Report

Recurrent Pseudomonas Infection Associated with Neutrophil Dysfunction ANN 0. SHIGEOKA and HARRY R. HILL’

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From the Division of Clinical Immunology, Department of Pediatrics and the Department of Pathology, University of Utah College of Medicine, Salt Lake City, Utah, USA

ABSTRACT. A 72-year-old male is described with a history of 4 episodes of Pseudomonas aeruginosa sepsis and chronic otitis media caused by pseudomonas species. In vitro testing of the patient’s polymorphonuclear leukocytes (PMNs) revealed profoundly abnormal chemotactic responses and defective intracellular killing of ps. aeruginosa, Staphylococcus aureus and Escherichia coli. Chemiluminescence production by the patient’s PMNs in response to opsonized zymosan as well as endotoxin stimulated nitroblue tetrazolium dye reduction were markedly depressed. These data indicate the presence of a profound, apparently acquired, defect in PMN function in an elderly male. Detailed evaluation of adult patients with recurrent infections may reveal similar, apparently acquired defects in PMN function.

INTRODUCTION The majority of abnormalities of polymorphonuclear leukocyte (PMN) function usually present with recurrent infections in childhood and many are hereditary in nature (6, 16, 17). Metabolic abnormalities of the PMN resulting in defective intracellular microbicidal mechanisms clinically evident as chronic granulomatous disease (CGD) are the best described of these disorders (17). Acquired chemotactic, phagocytic and bactericidal PMN dysfunction may occur secondary to other systemic diseases such as diabetes mellitus (10, 13), rheumatoid arthritis (14) or acute myelogenous leukemia (1). Solberg et al. (19, 22) have even observed defective PMN bactericidal activity in patients with the early or “preleukemic” phase of acute myelomonocytic or myeloblastic leukemia. Overwhelming infection resulting in toxic changes in PMNs may also produce functional abnormalities (1 1, 21) as will certain antibiotics such as tetracycline ( 5 ) . The elderly patient reported here had recent onset of recurrent severe pseudomonas infections associated with defects in PMN chemotaxis, chemiluminescence and intracellular killing of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Acquired defects in PMN function may be responsible for the recurrent infection suffered by some previously healthy adult patients. The assessment of such patients should

include a careful screen of neutrophil granulocyte function. CASEREPORT A 72-year-old white male was hospitalized in September 1976, with fever and acute respiratory distress accompanied by acute radiologic changes on chest films compatible with pneumonia. He had no history of significant infections until 1970. At that time a left lower lobe pneumonia and pleural effusion were present although no positive cultures were obtained. In 1972 and again in March 1976, he had episodes of Ps. aeruginosa sepsis with pneumonia (left and right lower lobe pneumonias respectively). During that time he was treated for chronic otitis media from which a pseudomonas species was consistently cultured despite local and systemic therapy. At the time of the last admission, blood cultures were negative. A pseudomonas organism, which was not further identified, was isolated from ear, throat and urine cultures. Bone marrow biopsy and skeletal survey did not indicate invasive marrow disease. Serum studies showed the presence of K and A chains of polyclonal origin, but no evidence of myeloma. Therapy with carbenicillin and amikacin resulted in slow defervescence and improvement of his pneumonia over a 10-day period. His course was complicated by mild congestive heart failure, gastrointestinal bleeding on the 4th day of hospitalization, and confusion developing 15 days after admission. On the 18th day of hospitalization the patient expired following progressive central nervous system impairment and increased respiratory distress associated with wheezing and diffuse rales.

Dr Hill is an investigator of the Howard Hughes Medical Institute.

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PATIENT WITH PSEUDOMONAS INFECTION

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PATIENTS WITH BACTERIAL INFECTION

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Fig. 1. Chemotactic indices of the patient with

pseudomonas infection in comparison with control patients and patients with active bacterial infection.

\\ Autopsy revealed a right lower lobe inflammatory process without identifiable microorganisms. Brain and spinal cord findings were unremarkable. No evidence of malignancy was present; lymph nodes showed normal architecture. Bone marrow examination, however, revealed a marked increase in mature histiocytic cells, a minimal increase in plasma cells and myeloid elements with decreased numbers of megakaryocytes. The spleen also showed mature histiocytic infiltrates with moderate erythrophagocytosis. Special stains revealed no increase in reticulum fibers and no evidence of any microorganisms. It was felt that a benign granulomatous process was present in addition to the pulmonary pathology.

MATERIALS AND METHODS Chemotaxis. Chemotaxis was studied by a micropore filter technique described in detail elsewhere (7, 9). Leukocyte rich plasma was obtained by gravity sedimentation from heparinized blood and adjusted to a neutrophil concentration ranging from 5 X lo6 to 15X 1P ml in medium 199 (MicrobiologicalAssociates, Inc., Bethesda, Md.). 0.1 ml of this suspension diluted to 0.4 ml in medium 199 was deposited on one side of a 5 p Millipore filter by cytocentrifugation. Filters were placed in modified Boyden chambers and the chemotactic stimulus added to the attractant side. Following incubation for 3 h at 37°C filters were removed and stained. A chemotactic index was calculated by dividing the number of PMN migrating completely through the filter by the total number of neutrophils delivered to the starting side of the filter (9). PMN random mobility was assessed by determining the chemotactic index when no chemotactic gradient was present in the modified Boyden chamber. Scund J Infect Dis 10

CONTROL PMNs

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Fig.2. Bactericidal assays for Staph. aureus 502A, E. coli, and Ps. aeruginosa.

Nitroblue tetrazolium (NBT) test. NBT dye reduction by PMN was determined by a modification of the method of Park et al. (15). Equal volumes of heparinized blood and 0.1% NBT solution were incubated for 30 min at 37°C and the percentage of PMN containing black deposits of formazan determined. Phagocytic and bactericidal capacity. The in vitro phagocytic and bactericidal capacity of neutrophils for E. coli, Staph. aureus, and Ps. aeruginosa was studied by the method of Quie et al. (17) utilizing normal adult serum as opsonin for the organism, and a ratio of one PMN to one bacteria. Samples were evaluated at 0, 60, and 120 min from both supernatant and cell fractions. In the same test system patient serum was compared with control adult serum for opsonic activity utilizing normal adult PMN as the phagocyte. Chemiluminescence (CL). PMN function was also evaluated by a CL procedure previously described (8). Opsonized zymosan particles were prepared by incubating zymosan in normal adult serum in a concentration of 10 mg zymosan to 1 ml serum. This mixture was rotated for 1 h at 37°C. Leukocyte rich plasma was obtained as described above, washed twice in sterile PBS and adjusted to a concentration of 1.OX lo7 PMN/ml. Preopsonized zymosan resuspended in PBS (0.2 ml of a 10 mglml suspension) was added to 0.5 ml of the leukocyte suspension (1 x lo7

Acquired PMN defects

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PMN CHEMILUMINESCENCE PRODUCED BY PATIENT WITH RECURRENT pSEUDOMONAS INFECTIONS

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PMN/ml) and 2.8 ml PBS in a Poly Q scintillation vial (Beckman, Fullerton, Calif.) which had been wrapped in foil and stored in the dark for at least 18 h prior to use. Each mixture was immediately counted in a Beckman LS 100 liquid scintillation counter, with one photomultiplier tube disconnected. Each sample was counted for 1 min at approximately 10 min intervals for 90 min and CL expressed as counts per min.

RESULTS The patient’s neutrophil granulocytes were evaluated during his last admission while ill and presumably infected. He was being treated at the time with carbenicillin and amikacin. In spite of his infection, there was no evidence of vacuolization, toxic granulations or Dohle bodies in the peripheral blood granulocytes. A profound defect in PMN chemotaxis was observed 6 days and 13 days from admission (Fig. 1). The patient’s chemotactic indexes (16 and 17) were profoundly depressed when

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Fig. 3. Neutrophil CL elicited over a 90 min period by opsonized zymosan. The patients’ PMNs and CGD-PMNs are compared to normal adult PMNs.

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compared to those of healthy controls. The defect in chemotaxis is even more apparent, however, when compared to the chemotactic responsiveness of otherwise normal patients with acute bacterial infections (7). Random mobility of the patient’s PMNs was not different from controls. The patient’s PMNs were also defective in bactericidal activity against Staph. aureus, E. cob, and Ps. aeruginosa opsonized in normal adult serum (Fig. 2). Spontaneous NBT dye reduction occurred in 4% of the patient’s PMNs (normal 4+3 %). Following stimulation with E. coli endotoxin this rose to 9% in the patient’s cells versus 36% in a simultaneously tested control. Myeloperoxidase staining of the patient’s PMN indicated normal myeloperoxidase content. The patient and control PMNs were also evaluated for their ability to produce CL. Upon exposure of normal cells to opsonized zymosan a marked peak in C L was observed (Fig. 3). In contrast, the patient’s cells produced no more C L than Scand J Infect Dis I0

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the mixture containing cells alone or that with CGD PMNs. Immunoglobulin and complement levels were as follows: IgG-8.7 g/l (normal 73-20), IgM-1.2 g/l (normal 0.6-2.5) and IgA-2.6 g/1 (normal 0.8-4.8), C3-0.8 g/l (normal 03-13), C4-0.17 g/l (normal 0.2-0.5) and C3PA-0.12 g/l (normal 0.12-0.3). The patient’s serum or plasma did not contain cell directed inhibitors of chemotaxis like those previously described (2, 20). In addition, an increased concentration of the chemotactic factor inactivator described by Ward and Bereberg (24) was not detected. The patient’s serum contained normal opsonins for Staph. aureus, E. coli, and Ps. aeruginosa. DISCUSSION The patient presented in this report was an elderly man with late onset of recurrent severe pseudomonas infections. He was found to have defective PMN chemotaxis and deficient bactericidal activity. The magnitude of the defect in killing as well as the CL response of his PMNs appeared similar to that in CGD; however, his age at onset would support an acquired rather than primary abnormality in function. Young and Armstrong (25) have demonstrated that human immunity to pseudomonas infection is most likely dependent upon heat stable and heat labile opsonins as well as normally functioning granulocytes. They point out that patients with leukemia and severe burns who are at increased risk to develop systemic pseudomonas infections have been shown to have either a lack of granulocytes or abnormally functioning ones. In addition McEuen et al. (12) have shown that different pseudomonas strains may have increased virulence in association with resistance to phagocytosis. Increasing the bacteria to phagocyte ratio makes the defect more apparent with these virulent strains suggesting the possibility that pseudomonas organisms may directly or indirectly alter phagocyte function. At the time of leukocyte testing, our patient did not have systemic pseudomonas infection, however, making this an unlikely explanation for his PMN abnormality. Additional case reports have described possible acquired PMN chemotactic and bactericidal defects. Edelson et al. (3) described a 24-year-old woman with recurrent gram-negative infections who, like our patient, had chemotactic and bactericidal abnormalities. A 65-year-old man (23) had late

onset of Staph. aureus infection with defective phagocytosis and killing of this organism but normal PMN myeloperoxidase, HMP shunt activity and NBT test. Familial lipochrome histiocytosis in 3 sisters (4) parallels our patient’s history in some respects. These women suffered from recurrent infections, primarily pneumonias, but also had arthritis, splenomegaly and lipochrome pigmentation in histiocytes. PMN studies (18) indicated a CGD-like defect, with depressed resting HMP shunt activity and O2 consumption, although NBT tests showed some dye reduction. The present patient, as well as those mentioned above, suggest that acquired neutrophil defects may be more common in adults than previously recognized. The laboratory evaluation of such patients should include some measure of the ability of their PMNs to chemotax, phagocytize and kill microorganisms. Further delineation of the mechanisms resulting in these acquired phagocyte abnormalities should contribute significantly to our understanding of the biochemistry and function of the neutrophil. ACKNOWLEDGEMENT This work was supported, in part, by U.S. Public Health Service Grant A1 13150 and AM21 140 and a grant from the Trasher Foundation.

REFERENCES 1. Davis, H. T., Brunning, R. D. & Quie, P. G.: Polymorphonuclear leukocyte myeloperoxidase deficiency in a patient with myelomonocytic leukemia. N Engl J Med 285: 789, 1971. 2. Demeo, A. N . & Anderson, B. R.: Defective chemotaxis associated with a serum inhibitor in cirrhotic patients. N Engl J Med 286: 735, 1972. 3. Edelson, P. J., Stites, D. P., Gold, S. & Fudenberg, H. H.: Disorders of neutrophil function, defects in the early stages of the phagocytic process. Clin Exp Immunol 13: 21, 1973. 4. Ford, D. K., Price, G. E., Culling, C. F. A. & Vassar, P. S.: Familial lipochrome pigmentation of histiocytes with hyperglobulinemia, pulmonary infiltration, sdlenomegaly, and susceptibility t o infection. Am J Med 33: 478, 1%2. 5. Forsgren, A., Schmeling, D. & Quie, P. G.: Effect of tetracycline on the phagocyte function of human leukocytes. J Infect Dis 130: 412, 1974. 6. Gray, G. R., Klebanoff, S. J., Stamatoyannopoulos, G., Austin, T., Naiman, S. C., Yoshida, A., Kliman, M. R. &Robinson, G. C. F.: Neutrophil dysfunction, chronic granulomatous disease, and nonspherocytic haemolytic anaemia caused by complete deficiency of glucose-6-phosphate dehydrogenase. Lancet 2: 530, 1913.

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Acquired PMN defects 7. Hill, H. R., Gerrard, J., Hogan, N. A. & Quie, P. G.: Hyperactivity of neutrophil leukotactic responses during active bacterial infection. J Clin Invest 53: 9%, 1974. 8. Hill, H. R., Hogan, N. A., Bale, J. F. & Hemming, V. G.: Evaluation of nonspecific (alternative pathway) opsonic activity by neutrophil chemiluminescence. Int Arch Allergy Appl Immunol53: 490, 1977. 9. Hill, H. R., Hogan, N. A., Mitchell, T. G. & Quie, P. G.: Evaluation of a cytocentrifuge method for measuring neutrophil granulocyte chemotaxis. J Lab Clin Med 86: 703, 1975. 10. Hill, H. R., Sauls, H. A., Dettloff, J. L. & Quie, P. G.: Impaired leukotactic responsiveness in patients with juvenile diabetes mellitus. Clin Immunol Imrnunopathol2: 395, 1974. 1 1 . McCall, C. E., Caves, J., Cooper, R. & DeChatelet, L.: Functional characteristics of human toxic neutrophils. J Infect Dis 124:68, 1971. 12. McEuen, D. D., Blair, P., Delbene, V. E. & Eurenius, K.: Correlation between pseudomonas bum wound infection and granulocyte antibacterial activity. Infect Immun 13: 1360, 1976. 13. Mowat, A. G. & Baum, J.: Chemotaxis of polymorphonuclear leukocytes from patients with diabetes mellitus. N Engl J Med 284: 621, 1971. 14. - Chemotaxis of polymorphonuclear leukocytes from patients with rheumatoid arthritis. J. Clin Invest 50: 2541, 1971. 15. Park, B. H., Fikrig, S. M. & Smithwick, E. M.: Infection and nitroblue-tetrazoleum reduction by neutrophils. Lancet 2: 532, 1968. 16. Quie, P. G., Kaplan, E. L., Page, A. R., Gruskay, F. L. & Malawista, S. E.: Defective polymorphonuclear-leukocyte function and chronic granulomatous disease in two female children. N Engl J Med 278: 976, 1968. 17. Quie, P. G., White, J. G., Holmes, B. & Good R. A.: In vitro bactericidal capacity of human polyrnorphonuclear leukocytes: Diminished activity in chronic granulomatous disease. J Clin Invest 46: 668, 1967. 18. Rodey, G. E., Park, B. H., Ford, D. K., Gray, B. H. & Good, R. A.: Defective bactericidal activity of peripheral blood leukocytes in lipochrome histiocytosis. Am J Med 49: 322, 1970. 19. Schreiner, A. & Solberg, C. 0.: Neutrophil dysfunction and granulomatosis in the preleukemic state. Scand J Infect Dis 8: 53, 1976. 20. Smith, C. W., Hollers, J. C., Dupree, E., Goldman, A. S. & Lord, R. A.: A serum inhibitor of leukotaxis in a child with recurrent infections. J Lab Clin Med 79: 875, 1972. 21. Solberg, C. 0. & Hellum, K. B.: Neutrophil granulocyte function in bacterial infections. Lancet 2: 727, 1972. 22. Solberg, C. O., Schreiner, A., Hellum, K. B. & Hamre, E.: Neutrophil granulocyte function in the early diagnosis of acute myelomonocytic and myeloblastic leukaemia. Acta Med Scand 197: 147, 1975. 23. Tan, J. S., Straws, R. G., Akabutu, J., Kauffman, C.

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A., Mauer, A. M. & Phair, J. P.: Persistent neutrophil dysfunction in an adult, combined defect in chemotaxis, phagocytosis and intracellular killing. Am J Med 57: 251, 1974. 24. Ward, P. A. &Bereberg, J. L.: Defective regulation of inflammatory mediators in Hodgkin’s disease. N Engl J Med 290: 76, 1974. 25. Young, L. S. & Armstrong, D.: Human immunity to Pseudomonas aeruginosa. I. In-vitro interaction of bacteria, polymorphonuclear leukocytes, and serum factors. J Infect Dis 126: 257, 1972. Address for reprints: A . Shigeoka, M.D., University of Utah Medical Center, Department of Pediatrics, Salt Lake City, Utah 84132, USA

Scutrd J Itfect Dis 10

Recurrent Pseudomonas infection associated with neutrophil dysfunction.

Scandinavian Journal of Infectious Diseases ISSN: 0036-5548 (Print) 1651-1980 (Online) Journal homepage: http://www.tandfonline.com/loi/infd19 Recur...
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