The Oncologist, Vol. 2, No. 2, 121–124,
April 1997
© 1997 AlphaMed Press
SPECIAL SECTION
PHYSICIAN EDUCATION |
Apoptosis and its Role in Neutrophil Dysfunction in AIDS
David L. Pitrak
University of Illinois, Department of Medicine, Infectious Diseases, Chicago, Illinois, USA and West Side VA Medical Center, Chicago, Illinois, USA
Correspondence:
David L. Pitrak, M.D., West Side VA Medical Center, 820 South Damen Avenue, M/P 111, Chicago, Illinois 60612, USA. Telephone: 312-666-6500, extension 3457; Fax: 312-455-5893.
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INTRODUCTION
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Apoptosis, or programmed cell death, is a mechanism of cell death in many biological processes, including growth and development, normal cell turnover, and tissue remodeling. Apoptosis is also very important in the regulation of immune responses. Apoptosis of B cells and T cells is key in regulating humoral and cell-mediated immune responses. Dysregulation and interruption of apoptosis is involved in the clonal proliferation of malignant cells. If pharmaceutical agents could be developed that trigger apoptosis rather than cause tumor necrosis, substantial toxicity could be avoided in cancer treatment. There are other implications for the oncologist as well. Accelerated apoptosis may have significant consequences for the immune system; cancer patients are at risk for infectious complication. One condition where apoptosis seems to have an adverse effect on the immune system is the acquired immune deficiency syndrome (AIDS). There is considerable evidence that apoptotic cell death significantly contributes to the depletion and dysfunction of CD4+ lymphocytes in AIDS, including cells not infected with the human immunodeficiency virus (HIV).
Neutrophils undergo apoptosis, and, in fact, are released from the bone marrow programmed to die within 24 hours. The apoptotic neutrophils are removed efficiently from the circulation by mononuclear phagocytes. Neutrophil apoptosis, like apoptosis of other cell types, can be affected by a variety of exogenous factors, so the process can be accelerated or decelerated. This can effect the numbers of circulating neutrophils and the function of these cells. We were interested to examine whether accelerated neutrophil apoptosis may contribute to HIV-related neutropenia and neutrophil dysfunction, similar to the depletion and dysfunction of T-helper cells. This is an area of interest to us because of the wide variety of bacterial infections that complicate the course of HIV disease.
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INFECTIONS AND NEUTROPENIA IN PATIENTS WITH AIDS OR HIV INFECTION
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Although opportunistic infections due to depressed cell-mediated immunity are the hallmark of AIDS, infections with common bacterial pathogens also occur with increased frequency and severity in HIV-infected patients [1–3]. These infections may be refractory to therapy, and recurrences are a significant problem. Some centers have seen serious bacterial infections become the single most common cause of HIV-related morbidity and mortality [4, 5]. Many of the pathogens isolated from HIV-infected patients are associated with abnormalities of humoral immunity, neutropenia, or neutrophil dysfunction. For example, serious infections with Pseudomonas aeruginosa are frequent in advanced HIV infection, including community-acquired infections in non-neutropenic patients without other classic risk factors for Pseudomonas infections. Neutrophils are known to be the major effector cells in host defense against this pathogen, and the occurrence of Pseudomonas infections in non-neutropenic patients supports the hypothesis that there is significant impairment of neutrophil function in advanced HIV infection.
Neutropenia is a frequent complication of HIV infection, although absolute neutropenia with counts <0.5 x 109/l is infrequent unless the patient is receiving bone marrow-suppressive medications [6]. Neutropenia is a significant risk factor for bacterial infections in HIV-infected patients. In addition to neutropenia, varieties of qualitative defects in neutrophil function have been described in HIV infection, including abnormal chemotaxis, phagocytosis, oxidative metabolism, and bacterial killing [7, 8]. The single most important clinical parameter predicting neutrophil dysfunction is the stage of HIV infection as determined by absolute CD4+ lymphocyte count, with the greatest degree of impairment seen in patients with CD4+ <100/µl [8]. There is an inverse correlation between absolute neutrophil count (ANC) and CD4+ lymphocyte count, but functional impairment can occur even in patients without neutropenia.
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NEUTROPHIL ACTIVATION
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In vivo activation of neutrophils has been reported. Elbim and coworkers [9] have noted increased surface expression of CD11b/CD18, reduced expression of L-selectin, increased actin polymerization, and increased spontaneous H202 production, all evidence for activation, using flow cytometric analysis of neutrophils in whole blood from HIV-infected patients [9]. Our lab has demonstrated an increase in the ratio of unprimed to primed neutrophil chemiluminescence responses in HIV-infected patients in a whole blood assay [10]. This increase in unprimed responses relative to the maximal primed responses indicates that the neutrophils are being activated or primed in vivo, and this in vivo activation occurs at all stages of disease and in the absence of any secondary infectious complication. Patients with advanced HIV disease have a significant decrease in maximal primed chemiluminescence with a relatively high unprimed response. In vivo activation may contribute to the inability of the neutrophils to respond to a second stimulus, such as a bacterial pathogen.
Neutrophil activation, however, is not the only mechanism involved in neutrophil dysfunction. It is known that neutrophils undergoing apoptosis have impaired function, and we have demonstrated that neutrophil apoptosis is markedly accelerated in patients with AIDS [11]. Neutrophils were isolated from normal subjects and from patients with advanced HIV infection (CD4+ <200/µl) and cultured for 18 hours. At different time points, cells were examined for morphological changes of apoptosis by light microscopic examination of Wright-stained cytospin preparations and by electron microscopy. Neither the neutrophils from control subjects nor those from patients showed evidence of apoptosis just after isolation, but over time, the progressive cytoplasmic shrinkage and nuclear condensation characteristic of apoptosis could be observed. Clearly, a higher proportion of neutrophils from the patients with AIDS showed advanced apoptosis after 18 hours in culture (Fig. 1A to 1F
). The rate of apoptosis can be quantitated after staining with acridine orange and ethidium bromide, fluorescent dyes that intercalate DNA. Flow cytometry also was used to demonstrate apoptosis after fluorescent staining with propidium iodine, another fluorescent dye that intercalates DNA. Finally, DNA was isolated from neutrophils collected from patients and normal subjects and subjected to electrophoresis on agarose gels to observe the DNA fragmentation or "laddering" characteristic of apoptosis. Neutrophils isolated from patients with AIDS show the morphological changes of apoptosis and DNA fragmentation much more rapidly relative to controls at all time points (3, 6, and 18 hours) in culture (Fig. 2). The morphological findings on microscopy were confirmed by flow cytometry (Fig. 3) and DNA electrophoresis. This acceleration of apoptosis is associated with a decrease in viability after 18 hours. Accelerated apoptosis is likely to contribute to the neutrophil dysfunction seen in these patients.
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Figure 1. Morphological changes of neutrophil apoptosis. Cytospins of neutrophils in culture were treated with Wright’s stain and examined with a light microscope. A) Neutrophils from a control subject at 0 h in culture show normal morphology; neutrophils from patients with AIDS (not shown) also show normal morphology at 0 h; light microscopy. B) After 18 h in culture, apoptotic cells with decreased cytoplasm and pyknotic nuclei can be identified in the control subject culture; light microscopy. C) After 18 h in culture, almost all the neutrophils from a patient with advanced HIV infection are apoptotic; light microscopy. D) Control neutrophils at 0 h; transmission electron microscopy. E) Control neutrophils at 18 h; transmission electron microscopy. F) Neutrophils from a patient with AIDS after 18 h in culture; transmission electron microscopy. (Adapted from [11]; used with permission.)
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Figure 2. Neutrophils isolated from patients with AIDS have increased apoptosis (panel A) and decreased viability (panel B) compared with those from control subjects. Solid squares, AIDS patients (n=10); solid circles, control subjects (n=7). (Adapted from [11]; used with permission.)
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Figure 3. Apoptosis is increased in AIDS, demonstrated by flow cytometry after staining with propridium iodide (PI). (Adapted from [11]; used with permission.)
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The mechanism(s) for accelerated apoptosis is not known. Proinflammatory cytokines and other inflammatory mediators can induce apoptosis, and the absence of other cytokines or growth factors can promote apoptosis. Granulocyte colony-stimulating factor (G-CSF) is one growth factor known to decrease the rate of neutrophil apoptosis and prolong the survival of neutrophils in vitro. There is increased neutrophil survival due to decreased apoptosis ex vivo during administration of recombinant human G-CSF (Filgrastim). We have examined the effects of Filgrastim administration to AIDS patients on ex vivo neutrophil apoptosis. Neutrophils isolated from patients receiving Filgrastim 300 µg subcutaneously daily or every other day for seven days showed a significant decrease in the rate of apoptosis while on therapy [12]. We have recently completed a trial examining the effects of Filgrastim on neutrophil function in HIV infection, and the results show significant increases in neutrophil oxidative capacity and bacterial killing, even in patients with advanced disease who had significantly impaired function before treatment (Fig. 4 and Fig. 5) [13]. This improvement in function correlated with and may be explained by the reversal of accelerated apoptosis.
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Figure 4. This graph represents the chemiluminescence (CL) response to zymosan opsonized with human complement (hc-OPZ) after priming with platelet-activating factor. Priming results in maximal opsonin receptor expression (MORE) by the neutrophils compared with neutrophils with circulating opsonin receptor expression (CORE). The results here are for patients with HIV infection at different stages of infection before, during, and after therapy with Filgrastim relative to control subjects and high-risk control patients.
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Figure 5. This graph shows the percent killing (colony-forming units (CFU) per milliliter without neutrophils) of serum-resistant Escherichia coli opsonized with 10% pooled normal human serum. Data are shown for the HIV-infected patient groups before, during, and after Filgrastim treatment compared with the control groups.
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IMPLICATIONS FOR THE ONCOLOGY SETTING
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These studies suggest that accelerated neutrophil apoptosis and neutrophil activation result in impaired neutrophil function, contributing to the risk of bacterial infections in patients infected with HIV. Although we are unaware of reports of any other disease or condition associated with accelerated neutrophil apoptosis, it is likely that accelerated neutrophil apoptosis occurs in other diseases associated with an increase in bacterial infection. Accelerated apoptosis of immune system effector cells may contribute to the risk of bacterial and opportunistic infections in other disease states, including malignancies. Furthermore, radiation therapy and chemotherapy may cause immune dysfunction by accelerating apoptotic cell death of cells of the immune system. For example, it is well established that ionizing radiation caused lymphocytes to die via apoptosis.
Cytokines and growth factors can interrupt apoptosis. Therapy with Filgrastim decreases the rates of neutrophil apoptosis and improves neutrophil function in HIV infection, and is potentially beneficial in the prevention and/or therapy of secondary bacterial infections that afflict these patients. Certainly clinical trials are warranted at this time. Filgrastim therapy has potential benefit in other clinical situations, such as patients with cancer receiving radiation or chemotherapy, not only in patients with neutropenia, but also non-neutropenic patients with neutrophil dysfunction.
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REFERENCES
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- Witt DJ, Craven DE, McCabe WR. Bacterial infections in adult patients with the acquired immune deficiency syndrome (AIDS) and AIDS-related complex. Am J Med
1987;82:900–906.[Medline]
- Nichols L, Balogh K, Silverman M. Bacterial infections in the acquired immune deficiency syndrome. Am J Clin Pathol
1989;92:787–790.[Medline]
- Kielhofneer M, Atmar RL, Hamill RJ et al. Life-threatening Pseudomonas aeruginosa infections with human immunodeficiency virus infection. Clin Infect Dis
1992;14:403–411.[Medline]
- Barat LM, Gunn JE, Steger KA et al. Causes of fever in patients infected with human immunodeficiency virus who were admitted to Boston City Hospital. Clin Infect Dis
1996;23:320–328.[Medline]
- Stein M, O’Sullivan P, Wachtel T et al. Causes of death in persons with human immunodeficiency virus infection. Am J Med
1992;93:387–390.[Medline]
- Zon LI, Groopman JE. Hematologic manifestations of the human immune deficiency virus (HIV). Semin Hematol
1988;25:208–218.[Medline]
- Ellis M, Gupta S, Galant S et al. Impaired neutrophil function in patients with AIDS or AIDS-related complex: a comprehensive evaluation. J Infect Dis
1988;158:1268–1276.[Medline]
- Pitrak DL, Bak PM, DeMarais P et al. Depressed neutrophil superoxide production in human immunodeficiency virus infection. J Infect Dis
1993;167:1406–1410.[Medline]
- Elbim C, Prevot MH, Bouscarat F et al. Polymorphonuclear neutrophils from human immunodeficiency virus-infected patients show enhanced activation, diminished fMLP-induced L-selectin shedding, and an impaired oxidative burst after cytokine priming. Blood
1994;84:2759–2766.[Abstract/Free Full Text]
- Mullane K, Pitrak D, Bilek M et al. In vivo neutrophil activation and burnout in HIV infection. Clin Res
1994;42:155a.
- Pitrak DL, Tsai HC, Mullane KM et al. Accelerated neutrophil apoptosis in the acquired immunodeficiency syndrome. J Clin Invest
1996;98:2714–2719.[Medline]
- Pitrak DL, Sutton SH, Tsai HC et al. Accelerated neutrophil apoptosis in AIDS: Partial reversal with r-metHuG-CSF. Proceedings of 33rd Annual Meeting of the Infectious Diseases Society of America
1995;508a.
- Pitrak DL, Mullane KM, Bilek M et al. Filgrastim (r-metHuG-CSF) treatment of HIV-infected patients improves neutrophil function. Proceedings of the XI International Conference on AIDS. 1996:Th.B 4181.
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Introduction
Infections and Neutropenia in...
