Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0205072 on April 27, 2005

Published online before print April 27, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0205072v1
78/2/338    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Spellberg, B. J.
Right arrow Articles by Ibrahim, A. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Spellberg, B. J.
Right arrow Articles by Ibrahim, A. S.
(Journal of Leukocyte Biology. 2005;78:338-344.)
© 2005 by Society for Leukocyte Biology

A phagocytic cell line markedly improves survival of infected neutropenic mice

Brad J. Spellberg*,{dagger},1, Mary Collins*, Samuel W. French{dagger},{ddagger}, John E. Edwards, Jr*,{dagger}, Yue Fu*,{dagger} and Ashraf S. Ibrahim*,{dagger}

* Division of Infectious Diseases and
{ddagger} Department of Pathology, Harbor-University of California at Los Angeles Medical Center; and
{dagger} The David Geffen School of Medicine at the University of California at Los Angeles

1 Correspondence: Division of Infectious Diseases, Harbor-UCLA Medical Center, 1124 West Carson St., Torrance, CA 90502. E-mail: bspellberg{at}labiomed.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Disseminated candidiasis is a frequent infection in neutropenic patients, in whom it causes 50% mortality, despite antifungal therapy. As the duration of neutropenia is the strongest predictor of survival in neutropenic patients with invasive fungal infections, neutrophil transfusions are a logical, therapeutic option. However, significant technical barriers have prevented the clinical use of neutrophil transfusions. To overcome these barriers, we identified a human phagocytic cell line that could be administered to candidemic hosts in lieu of freshly harvested neutrophils. HL-60 cells killed Candida albicans in vitro. Activation of HL-60 cells with dimethyl sulfoxide and retinoic acid abrogated the cells’ proliferation and augmented their killing of C. albicans. Administration of activated HL-60 cells to candidemic, neutropenic mice significantly improved survival (53% vs. 0%). Live HL-60 cells chemotaxed to sites of infection, phagocytized C. albicans, and reduced the fungal burden in key target organs. Although unactivated HL-60 cells also reduced tissue fungal burden in vivo, they did not improve survival as a result of their toxicity in infected mice. In contrast, no toxicity as a result of activated HL-60 cells was observed at up to 2 months of follow-up. To our knowledge, this is the first description of a cell line-based immunotherapy for an infectious disease. With further refinements, activated HL-60 cells have the potential to overcome the technical barriers to neutrophil transfusions.

Key Words: HL-60 • immunotherapy • Candida albicans • white cell transfusions


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Candida is among the most common causes of infections in patients with cancer or neutropenia. Disseminated candidiasis occurs in 2–6% of such patients [1 2 3 4 5 6 ], causing an unacceptable 50% mortality, despite antifungal therapy [7 8 9 10 11 ]. Furthermore, the potential for Candida spp. to develop resistance to conventional antifungal therapies has fueled concern regarding the future ability to treat such infections [12 , 13 ].

As survival of neutropenic patients with hematogenously disseminated fungal infections is related linearly to patients’ granulocyte counts [14 , 15 ], exogenous replacement of phagocytes is of great therapeutic potential for these infections. Although neutrophil transfusions have shown promising results [16 , 17 ], they fell out of favor in the early 1980s as a result of several technical difficulties [18 19 20 21 ]. First, harvesting sufficient neutrophils to mediate a protective effect (≥1x1011 neutrophils/day in infected patients; ref. [22 ]) is difficult to achieve [21 ]. Second, ex vivo neutrophils rapidly undergo apoptosis and quickly lose their ability to chemotax to and kill microorganisms [21 ]. Of note, this loss of microbicidal activity is particularly severe for killing Candida as compared with smaller bacterial organisms [23 ]. Third, trace but clinically significant quantities of red blood cells, lymphocytes, platelets, and/or donor alloantibodies inevitably contaminate even the most pure neutrophil harvests [24 , 25 ]. Therefore, the donor pool is limited by the need to cross-match blood types, and neutropenic recipients may be at risk for graft-versus-host disease or cytomegalovirus infection from transfused lymphocytes.

To garner the therapeutic benefit of neutrophil transfusions but avoid the above technical obstacles, we investigated the potential for an immortal phagocytic cell line to protect neutropenic mice against disseminated candidiasis. The HL-60 cell line was originally obtained by leukapheresis of peripheral blood cells from a patient with acute promyelocytic leukemia [26 ] and has been maintained in culture without genetic manipulation since that time [American Type Culture Collection (ATCC), Manassa, VA, package insert]. A major advantage of HL-60 cells is that they can be activated to differentiate toward a mature neutrophil phenotype [27 ]. Here, we show that activated HL-60 cells markedly improved the survival of neutropenic mice with disseminated candidiasis and reduced tissue fungal burden in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture, activation, and harvesting of cells
Human neutrophils were harvested by Ficoll-Paque (Amersham Biosciences, Piscataway, NJ) centrifugation of blood donated from healthy volunteers per the manufacturer’s recommendation. HL-60, THP-1 (ATCC), and MonoMac-6 cells (courtesy of Dr. Judy Berliner, UCLA Center for the Health Sciences, Los Angeles, CA) were cultured at 37°C in 5% CO2 in RPMI 1640 supplemented with glutamine (Irvine Scientific, Santa Ana, CA), 10% fetal bovine serum (Gemini BioProducts, Woodland, CA), 1% penicillin, streptomycin, and glutamine (Gemini BioProducts), and 50 µM ß-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). HL-60 cells were activated by incubation in the presence of 1.3% (v/v) dimethyl sulfoxide (DMSO; Sigma-Aldrich) and/or 2.5 µM retinoic acid (RA; Sigma-Aldrich). In some experiments, activated HL-60 cells were killed by incubation for 3 days in 1% sodium azide (Sigma-Aldrich) in culture media. Prior to their administration to mice, trypan blue exclusion confirmed that azide treatment resulted in complete killing of activated HL-60 cells. For harvesting, cells were centrifuged at 500 g, washed in Hanks’ balanced salt solution (HBSS; Irvine Scientific), and resuspended at the appropriate concentration for intraperitoneal (i.p.) therapy. The conditioned media (CM) of activated HL-60 cell cultures was washed in parallel for use as placebo. To wash the CM, the media was centrifuged and decanted, and HBSS was added to the residua at an equivalent volume to the final HL-60 cell suspension.

Dr. William A. Fonzi (Georgetown University, Washington, DC) supplied Candida albicans SC5314 [28 ], a well-characterized, clinical isolate, which is highly virulent in animal models. The organism was serially passaged three times in yeast peptone dextrose broth (Difco, Detroit, MI) and washed twice with phosphate-buffered saline (PBS; Irvine Scientific) prior to infection. The infectious inoculum was prepared by counting in a hemacytometer.

In vitro killing, proliferation, and phagocytosis assays
To determine their candidacidal effect, 8 x 104 phagocytes were cocultured with 4 x 103 C. albicans (20:1 ratio) for 4 h at 37°C in RPMI + 10% pooled human serum (Sigma-Aldrich). The cultures were sonicated, serially diluted, overlaid with yeast potato dextrose agar (Difco), and incubated overnight at 37°C. Colony-forming units (CFUs) were counted to assess killing of C. albicans.

To quantify the in vitro proliferative capacity [29 30 31 ], HL-60 cells were cultured for 4 days with or without DMSO. The cells were split to a uniform density of 5 x 105 cells/ml and cultured for a further 3 days with or without DMSO and/or RA. On day 7, viable cells were counted by trypan blue exclusion and compared with the baseline cell count on day 4.

In vitro phagocytosis was performed by labeling the HL-60 cells with CellTrace seminaphthorhodafluor (SNARF)-1 by the method of Tokalov and Gutzeit [32 ]. Briefly, HL-60 cells were labeled in the presence of 10 µm SNARF-1 for 10 min at 37°C and then washed three times prior to use. C. albicans, which constitutively overexpresses green fluorescent protein (GFP), was a kind gift from Dr. Brendan Cormack (Johns Hopkins University, Baltimore, MD) [33 ]. The phagocytosis assay was performed as per the in vitro killing assay except using a 1:1 ratio of HL-60:C. albicans.

Imaging
Confocal microscopy was performed on a Leica TCS SP2 microscope (Leica Microsystems, Banncokburn, IL). Confocal images were constructed by stacking 0.5 µ optical sections along the z-axis.

Mice and survival experiments
Male Balb/c mice (20–25 g) were obtained from the National Cancer Institute (Frederick, MD). Mice were made neutropenic by a single i.p. injection of high-dose cyclophosphamide (230 mg/kg) [34 ]. In pilot studies in which peripheral blood leukocytes were counted manually, this treatment regimen resulted in pancytopenia from days 1–7 post-cyclophosphamide treatment (days 0–6 post-infection given infection on day 1 post-cyclophosphamide), and recovery of cell counts began on day 8 post-cyclophosphamide (day 7 post-infection). Mice were infected via the tail-vein with 0.2 ml nonpyrogenic PBS containing 5 x 104 blastoconidia of C. albicans ~32 h after cyclophosphamide injection, as described previously [28 ].

Treatments (i.p.) with phagocytes or placebo were administered 30–60 min after infection on day 0 and again, on days 1 and 3 (three doses) or 2, 4, and 6 (four doses) post-infection. Four doses of 1.5 x 107 cells/mouse (~7.5x108 cells/kg) were the maximum logistically achievable, given the constraints of growing sufficient cells in a tissue-culture incubator (~1200 ml cell culture/mouse was required given cells grown at a density of 5x105 cells/ml, with 90% reduction in cell counts during activation). Of note, this treatment dose is approximately half the 1.5 x 109 cells/kg dose of neutrophils thought to be protective in humans [22 ]. Cell treatments were administered i.p. to avoid small vessel embolization resulting from an i.v. bolus of the thick cell suspension. All procedures involving mice were approved by the Institutional Animal Use and Care Committee, following the National Institutes of Health guidelines for animal housing and care.

Organ colony counts and histopathology
For determination of organ colony counts, mice were made neutropenic, infected, and treated with activated HL-60 cells or placebo on days 0, 2, and 4 post-infection. On day 5 post-infection, kidneys and brains were weighed, homogenized, serially diluted, plated on Sabouraud dextrose agar (Difco), and then incubated overnight at 37°C prior to counting CFUs [28 ].

For histopathology, mice were treated with three or four doses of HL-60 cells or control. Organs were harvested, fixed in zinc-buffered formalin followed by 80% ethanol, and then embedded in paraffin. Thin sections were cut and stained with periodic acid Schiff (PAS) or immunoperoxidase-labeled monoclonal antibody against proliferating cell nuclear antigen (PCNA; Signet Laboratories, Delhem, MA), a marker of active cell cycling, as described [35 ].

Statistics
Kaplan-Meier curves were compared pair-wise with the nonparametric log rank test. In vitro killing and replication and in vivo tissue fungal burden were compared with the nonparametric Steel test for multiple comparisons [36 ] or the Wilcoxon rank sum test, as appropriate. Results of survival studies performed on different days were pooled if the control curves were not statistically heterogenous by the Kolmogorov-Smirnov test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HL-60, but not other human phagocytic cell lines, killed C. albicans in vitro
Several human phagocytic cell lines were tested for their ability to kill C. albicans in vitro. HL-60 cells effectively killed C. albicans, whereas MonoMac-6 or THP-1 cells did not (Fig. 1A ). Furthermore, activation of HL-60 cells with DMSO and RA, which induces their maturation toward a neutrophil phenotype [37 , 38 ], significantly increased the candidacidal efficiency of HL-60 cells (Fig. 1A) . The maturation induced by DMSO and RA also abrogated HL-60 cell proliferation (Fig. 1B) . Based on these results, additional studies focused on HL-60 cells.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. Activation with DMSO and RA stimulates killing of C. albicans by HL-60 cells and inhibits HL-60 replication. (A) Median in vitro killing of C. albicans. Results are expressed as the percent reduction of CFUs from fungal-phagocyte cocultures versus simultaneous cultures containing C. albicans without phagocytes. Experiments were performed in triplicate and repeated three times. PMN, Polymorphonuclear neutrophils. (b) Median percent change relative to baseline counts. Experiments were performed in triplicate and repeated five times. *, P < 0.05, versus unactivated HL-60 cells; {ddagger}, P < 0.05, versus unactivated RA x3 days and DMSO x3 days. Error bars represent interquartile ranges.

 
Activated HL-60 cells protected neutropenic mice from disseminated candidiasis in a manner requiring HL-60 viability
Having demonstrated that activated HL-60 cells could kill C. albicans in vitro, their efficacy was tested in vivo in neutropenic, candidemic mice. Infected neutropenic mice were pooled and randomly sorted to receive treatment with 1.5 x 107 unactivated HL-60 cells, HL-60 cells activated with RA and DMSO, activated HL-60 cells killed with azide, placebo (washed supernatant from HL-60 cells activated with RA and DMSO), or THP-1 cells. Mice treated with live-activated or unactivated HL-60 cells displayed a marked reduction in signs consistent with infection, including ruffled fur and lethargy, compared with all other groups. However, starting on approximately day 6 post-infection, mice treated with unactivated HL-60 cells began to display increased ruffled fur and lethargy, compared with mice treated with live-activated HL-60 cells.

One hundred percent mortality by day 11 post-infection was observed in mice treated with placebo, THP-1 cells, unactivated HL-60 cells, or azide-killed, activated HL-60 cells (Fig. 2A ). Activated HL-60 cells significantly improved the survival of infected neutropenic mice versus all other groups (P<0.02 for all comparisons, Fig. 2A ). The improvement in survival was dose-dependent, as four doses of activated HL-60 cells resulted in 53% long-term survival, significantly greater than the 11% long-term survival seen in mice treated with three doses (P=0.02 by log rank test).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 2. Activated HL-60 cells markedly improve survival of neutropenic mice with disseminated candidiasis and enhance fungal clearance. (A) Survival for the three- or four-dose arms were censored at 35 or 60 days, respectively. N = 7 mice from one experiment for the killed-activated group. For other groups, n = 9 (unactivated), 10 (THP-1), 15 (for activated HL-60 cells), or 30 (placebo) mice from at least two experiments. (B) y-axes reflect lower limits of detection of CFU/g tissue. N = 16 mice per group from duplicate experiments. *, P < 0.05, versus placebo; {ddagger}, P < 0.05, versus all other groups. Error bars represent interquartile ranges.

 
HL-60 cells chemotaxed to sites of infection, phagocytized C. albicans in tissue, and reduced tissue fungal burden in vivo
To determine the mechanism of protection, we assessed CFUs of C. albicans in key target organs in mice treated with viable HL-60 cells versus controls. CFUs were determined after three treatment doses on day 5 of infection just prior to the first deaths in the placebo-treated mice. Consistent with previously published data [28 , 39 ], preliminary experiments confirmed that the kidney was the most heavily infected organ in this model and that brains were also heavily infected (data not shown). In contrast, livers and spleens were more lightly infected, so subsequent studies focused on the CFUs in kidneys and brains. Treatment with unactivated or activated HL-60 cells resulted in significant decreases in CFUs in the kidneys and brains (Fig. 2B) .

Histopathology performed on the day of neutrophil recovery (day 7 post-infection) demonstrated that brains and kidneys from mice treated with activated or unactivated HL-60 cells contained few foci of neutrophilic inflammation. These neutrophilic foci contained minimal if any visible fungal pseudohyphae (Fig. 3A ). In contrast, organs from mice treated with placebo or killed HL-60 cells contained numerous foci of infection containing extensively filamented hyphal mats (Fig. 3A) . Finally, PAS staining revealed cells morphologically consistent with leukemic blasts around the foci of infection only in mice treated with live HL-60 cells (Fig. 3A) . Immunohistochemistry for PCNA confirmed the presence of blast cells only in mice treated with live HL-60 cells (Fig. 3B) , indicative of chemotaxis of the leukemic cells from the peritoneum (where they were administered) to the sites of infection.



View larger version (139K):
[in this window]
[in a new window]
  		Figure 3. HL-60 cells chemotax to the site of infection and reduce tissue fungal burden in vivo. PAS (A)- or PCNA (B)-stained kidneys and brains from mice treated with placebo, killed-activated HL-60 cells, live-unactivated HL-60 cells, or live-activated HL-60 cells. PCNA+, Replicating cells stain brown. *, Cells morphologically consistent with leukemic blast cells seen in PAS-stained tissues. Original bar = 30 µm.

 
We hypothesized that HL-60 cells reduced tissue fungal burden by phagocytizing the C. albicans. To confirm their capacity to phagocytize the fungus in vitro, we coincubated activated HL-60 cells labeled with the fluorescent dye CellTrace SNARF-1 (Molecular Probes, Eugene, OR) and C. albicans expressing GFP [40 ]. Confocal microscopy and differential interference contrast (DIC) optics confirmed phagocytosis of the fungus by HL-60 cells in vitro (Fig. 4a and 4b ). To detect in vivo phagocytosis, neutropenic mice were infected and treated with activated HL-60 cells as done previously. However, histopathology was performed on day 5 (after three doses) rather than day 7 post-infection to detect earlier fungal lesions, prior to eradication of hyphae, thereby increasing the chance of visually detecting HL-60 cells and adjacent hyphae. Dual staining with PAS and PCNA confirmed phagocytosis of the fungus by HL-60 cells at multiple locations in the brain and kidney (Fig. 4c 4d 4e) .



View larger version (102K):
[in this window]
[in a new window]
  		Figure 4. Activated HL-60 cells phagocytize C. albicans in vitro and in vivo. Fluorescence (a) and DIC optics (b) confocal microscopy, demonstrating in vitro phagocytosis of C. albicans by activated HL-60 cells. Yellow indicates colocalization of red (SNARF-1-labeled HL-60 cells) and green (GFP-C. albicans) fluorescence. (c–e) In vivo phagocytosis demonstrated by dual PAS (pink C. albicans)-PCNA (brown HL-60 cells) staining of brain (c, original bar=10 µm; d, original bar=4 µm) and kidney (e, original bar=4 µm) from neutropenic, candidemic mice treated with three doses of activated HL-60 cells.

 
Unactivated, but not activated, HL-60 cells were toxic to infected neutropenic mice
As unactivated HL-60 cells reduced tissue fungal burden but did not improve survival in infected neutropenic mice, we tested their toxicity compared with activated HL-60 cells. Initially, uninfected neutropenic mice (n=8 per group) were treated i.p. with activated or unactivated HL-60 cells on days 1, 3, 5, and 7 post-cyclophosphamide therapy. None of the mice died. However, significant ascites developed in all mice the day after the first treatment and persisted until neutrophil recovery on day 8 post-cyclophosphamide, when the ascites resolved in all mice. As ascites had not been noted previously in any neutropenic-infected mice receiving HL-60 cell treatments, we hypothesized that systemic infection was required to induce HL-60 cell chemotaxis out of the peritoneal cavity. To evaluate the toxicity of the HL-60 cells in the presence of infection, neutropenic mice were infected with C. albicans as done previously and treated with four doses of unactivated HL-60 cells, activated HL-60 cells, or placebo (washed-activated HL-60 cell supernatant). To mitigate the effects of infection, each group was treated with liposomal amphotericin B (15 mg/kg/d i.v.) from days 2 through 5 post-infection. In contrast to the uninfected neutropenic mice, no ascites were observed in any of the infected neutropenic mice receiving HL-60 cells, supporting the hypothesis that systemic infection induced the cells to chemotax out of the peritoneum. None of the placebo-treated mice died, confirming the efficacy of liposomal amphotericin B in eradicating the infection. None of the mice treated with activated HL-60 cells died, confirming that activated cells were not toxic. However, half the mice treated with unactivated HL-60 cells died (P=0.025 vs. placebo and activated HL-60 cells, Fig. 5 ), confirming that unactivated HL-60 cells were toxic in the setting of infection.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 5. Unactivated HL-60 cells are toxic to mice, but activated HL-60 cells are not. N = 8 mice per group. Liposomal amphotericin B (LAmB) was dosed at 10 mg/kg i.v. daily from days 2 through 6 post-infection. *, P = 0.025, by log rank test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, this is the first demonstration of a cell line-based immunotherapy to treat an infection in vivo. Our results provide proof-of-principle of the potential for immortal phagocytic cells to recapitulate neutrophil functions in neutropenic hosts. We report that activated HL-60 cells markedly improved the survival of neutropenic mice infected with an otherwise 100% lethal inoculum of C. albicans. The lack of protection afforded by killed HL-60 cells and live THP-1 cells demonstrates that protection was a result of active HL-60 cell function and not a xenogeneic immune response to human antigens in the myeloablated mice.

Histopathology confirmed HL-60 cell phagocytosis of C. albicans in vivo in parenchymal organs. The reduction in tissue fungal burden in mice treated with viable HL-60 cells appeared significantly greater by histopathology than was reflected by the CFU data. These results are consistent with prior investigations [28 , 41 42 43 ], demonstrating that organ homogenization results in artificially low estimates of fungal burden in the presence of extensive filamentation (as seen in mice treated with placebo or killed HL-60 cells) but more accurately estimates fungal burden in the presence of blastoconidia or pseudohyphae (as seen in mice treated with viable HL-60 cells). Thus, the CFU results likely underestimated the efficacy of the HL-60 cells. Based on histopathology and CFU determinations, the mechanism of protection of HL-60 cells was reduction in tissue fungal burden.

It is surprising that unactivated HL-60 cells decreased kidney and brain fungal burden but did not improve survival. We therefore hypothesized that the unactivated HL-60 cells were toxic and resulted directly in the deaths of the infected animals. Indeed, unactivated HL-60 cells caused 50% mortality in infected, neutropenic mice. The mechanism of toxicity of unactivated HL-60 cells is under investigation. However, no toxicity of activated HL-60 cells was detected for up to 60 days post-infection. Therefore, a major therapeutic benefit of activation was to abrogate toxicity of HL-60 cells in infected neutropenic mice. These data emphasize the suitability of activated, but not unactivated, HL-60 cells for further preclinical investigation as a potential immunotherapy.

Activation also resulted in improved in vitro killing of C. albicans by HL-60 cells but did not diminish CFUs in vivo, likely because unactivated HL-60 cells have a higher proliferative capacity than activated HL-60 cells. Therefore, administration of the same dose of both cell types likely represents a higher functional dose of unactivated HL-60 cells than activated HL-60 cells, and reduction of tissue fungal burden represents a balance between HL-60 cell number and per-cell fungicidal activity.

It is not surprising that endogenous host factors are incapable of inducing "activation" of unactivated HL-60 cells. In the context of HL-60 cells, activation represents the induction of differentiation of HL-60 cells toward a mature neutrophil phenotype [27 ]. Thus, activation requires differentiation factors, such as DMSO and RA, which are distinct from what are normally thought of as activating factors for phagocytes, such as interferon-{gamma} [44 ]. In vivo authentic factors are incapable of inducing differentiation of leukemic cells. This is well known clinically; acute promyelocytic leukemia cells develop even in the presence of normal host signals but can be caused to differentiate in vivo with induction therapy with all trans RA. The potential for coadministration of unactivated HL-60 cells and RA to the mouse, as opposed to in vitro activation, is meritorious of further investigation.

A crucial consideration regarding the future potential use of activated HL-60 cells as an immunotherapy is the safety of the strategy. Several clinical trials have demonstrated the safety of transfusion of granulocytes from donors with chronic myelogenous leukemia into infected neutropenic recipients [45 46 47 ]. Although short-term engraftment of the leukemic cells occurred rarely in myeloablated recipients, the allogeneic leukemic cells were rejected once recipients’ immune systems recovered. More recently, a natural killer (NK) cell line has been administered as an anti-cancer strategy to patients in a phase I clinical trial, and again, no long-term engraftment or toxicity was seen [48 ]. These precedents establish that short-term infusion of allogeneic, immortal cells can be performed safely in humans. Finally, it is anticipated that any potential, future use of a phagocytic cell line-based immunotherapy would be limited to neutropenic patients infected with pathogens that do not respond well to available antimicrobial therapy. The unacceptably high mortality in such patients would make the risk to benefit the ratio of a cell line-based immunotherapy more favorable.

In summary, we describe a strategy for the propagation, activation, and administration of an immortal phagocytic cell line to infected, neutropenic mice. A variety of mechanisms to further enhance the activity and safety of activated HL-60 cells in neutropenic mice is under activate investigation. Possible safety mechanisms under investigation include stable integration of an inducible suicide trap in the immortal cells to allow purging of the cells’ subsequent host neutrophil recovery and irradiation of the cells, akin to the technique used in a recent clinical trial of immortal NK cells [48 ]. Future studies are planned to define the impact of such revisions to the immortal cells on the logistics of their in vitro growth and in vivo administration, their efficacy in vivo, and their lifespan in treated mice. Nevertheless, the results of the current study provide proof-of-principle of the potential for administered immortal cells to recapitulate phagocytic function in the setting of infected, neutropenic hosts.


   ACKNOWLEDGEMENTS
 
This work was supported by RO3 AI054531 (A. S. I.), RO1 AI19990 (J. E. E.), and KO8 AI060641 (B. J. S.) from the National Institute of Allergy and Infectious Diseases. J. E. E. is also supported by an unrestricted grant for Infectious Diseases from Bristol Myers Squibb. This study was conducted at the Los Angeles Biomedical Institute at Harbor-UCLA Medical Center. We thank Valentina Avanesian and Shalonda Ofoegbu for excellent technical assistance and Drs. Eric Brass and Michael Yeaman for helpful comments.

Received February 4, 2005; revised April 1, 2005; accepted April 5, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. el-Mahallawy, H. A., Attia, I., Ali-el-Din, N. H., Salem, A. E., Abo-el-Naga, S. (2002) A prospective study on fungal infection in children with cancer J. Med. Microbiol. 51,601-605[Abstract/Free Full Text]
  2. Hajjeh, R. A., Sofair, A. N., Harrison, L. H., Lyon, G. M., Arthington-Skaggs, B. A., Mirza, S. A., Phelan, M., Morgan, J., Lee-Yang, W., Ciblak, M. A., Benjamin, L. E., Sanza, L. T., Huie, S., Yeo, S. F., Brandt, M. E., Warnock, D. W. (2004) Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program J. Clin. Microbiol. 42,1519-1527[Abstract/Free Full Text]
  3. Krcmery, V., Jr, Oravcova, E., Spanik, S., Mrazova-Studena, M., Trupl, J., Kunova, A., Stopkova-Grey, K., Kukuckova, E., Krupova, I., Demitrovicova, A., Kralovicova, K. (1998) Nosocomial breakthrough fungaemia during antifungal prophylaxis or empirical antifungal therapy in 41 cancer patients receiving antineoplastic chemotherapy: analysis of aetiology risk factors and outcome J. Antimicrob. Chemother. 41,373-380[Abstract/Free Full Text]
  4. Martino, P., Girmenia, C., Venditti, M., Micozzi, A., Santilli, S., Burgio, V. L., Mandelli, F. (1989) Candida colonization and systemic infection in neutropenic patients. A retrospective study Cancer 64,2030-2034[CrossRef][Medline]
  5. Ribeiro, P., Sousa, A. B., Nunes, O., Aveiro, F., Fernandes, J. P., Gouveia, J. (1997) Candidemia in acute leukemia patients Support. Care Cancer 5,249-251[CrossRef][Medline]
  6. Safdar, A., Chaturvedi, V., Cross, E. W., Park, S., Bernard, E. M., Armstrong, D., Perlin, D. S. (2001) Prospective study of Candida species in patients at a comprehensive cancer center Antimicrob. Agents Chemother. 45,2129-2133[Abstract/Free Full Text]
  7. Wey, S. B., Mori, M., Pfaller, M. A., Woolson, R. F., Wenzel, R. P. (1988) Hospital-acquired candidemia. The attributable mortality and excess length of stay Arch. Intern. Med. 148,2642-2645[Abstract]
  8. Martino, P., Girmenia, C., Micozzi, A., Raccah, R., Gentile, G., Venditti, M., Mandelli, F. (1993) Fungemia in patients with leukemia Am. J. Med. Sci. 306,225-232[Medline]
  9. Safdar, A., Hanna, H. A., Boktour, M., Kontoyiannis, D. P., Hachem, R., Lichtiger, B., Freireich, E. J., Raad, I. I. (2004) Impact of high-dose granulocyte transfusions in patients with cancer with candidemia: retrospective case-control analysis of 491 episodes of Candida species bloodstream infections Cancer 101,2859-2865[CrossRef][Medline]
  10. Gudlaugsson, O., Gillespie, S., Lee, K., Vande Berg, J., Hu, J., Messer, S., Herwaldt, L., Pfaller, M., Diekema, D. (2003) Attributable mortality of nosocomial candidemia, revisited Clin. Infect. Dis. 37,1172-1177[CrossRef][Medline]
  11. Charles, P. E., Doise, J. M., Quenot, J. P., Aube, H., Dalle, F., Chavanet, P., Milesi, N., Aho, L. S., Portier, H., Blettery, B. (2003) Candidemia in critically ill patients: difference of outcome between medical and surgical patients Intensive Care Med. 29,2162-2169[CrossRef][Medline]
  12. Valentin, A., Le Guennec, R., Rodriguez, E., Reynes, J., Mallie, M., Bastide, J. M. (1996) Comparative resistance of Candida albicans clinical isolates to fluconazole and itraconazole in vitro and in vivo in a murine model Antimicrob. Agents Chemother. 40,1342-1345[Abstract]
  13. Kelly, S. L., Lamb, D. C., Kelly, D. E., Manning, N. J., Loeffler, J., Hebart, H., Schumacher, U., Einsele, H. (1997) Resistance to fluconazole and cross-resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol {delta}5,6-desaturation FEBS Lett. 400,80-82[CrossRef][Medline]
  14. Bodey, G. P., Buckley, M., Sathe, Y. S., Freireich, E. J. (1966) Quantitative relationships between circulating leukocytes and infection in patients with acute leukemia Ann. Intern. Med. 64,328-340
  15. Leibovici, L., Drucker, M., Samra, Z., Konisberger, H., Pitlik, S. D. (1995) Prognostic significance of the neutrophil count in immunocompetent patients with bacteraemia QJM 88,181-189
  16. Alavi, J. B., Root, R. K., Djerassi, I., Evans, A. E., Gluckman, S. J., MacGregor, R. R., Guerry, D., Schreiber, A. D., Shaw, J. M., Koch, P., Cooper, R. A. (1977) A randomized clinical trial of granulocyte transfusions for infection in acute leukemia N. Engl. J. Med. 296,706-711[Abstract]
  17. Herzig, R. H., Herzig, G. P., Graw, R. G., Jr, Bull, M. I., Ray, K. K. (1977) Successful granulocyte transfusion therapy for gram-negative septicemia. A prospectively randomized controlled study N. Engl. J. Med. 296,701-705[Abstract]
  18. Winston, D. J., Ho, W. G., Gale, R. P. (1980) Granulocyte transfusions Blood 56,324-326[Free Full Text]
  19. Strauss, R. G., Connett, J. E., Gale, R. P., Bloomfield, C. D., Herzig, G. P., McCullough, J., Maguire, L. C., Winston, D. J., Ho, W., Stump, D. C., Miller, W. V., Koepke, J. A. (1981) A controlled trial of prophylactic granulocyte transfusions during initial induction chemotherapy for acute myelogenous leukemia N. Engl. J. Med. 305,597-603[Abstract]
  20. Winston, D. J., Ho, W. G., Gale, R. P. (1982) Therapeutic granulocyte transfusions for documented infections. A controlled trial in ninety-five infectious granulocytopenic episodes Ann. Intern. Med. 97,509-515
  21. Huestis, D. W., Glasser, L. (1994) The neutrophil in transfusion medicine Transfusion 34,630-646[CrossRef][Medline]
  22. Marsh, J. C., Boggs, D. R., Cartwright, G. E., Wintrobe, M. M. (1967) Neutrophil kinetics in acute infection J. Clin. Invest. 46,1943-1953
  23. Glasser, L. (1977) Effect of storage on normal neutrophils collected by discontinuous-flow centrifugation leukapheresis Blood 50,1145-1150[Abstract/Free Full Text]
  24. Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 40-1998. A 49-year-old woman with thrombotic thrombocytopenic purpura and severe dyspnea during plasmapheresis and transfusion N. Engl. J. Med. 1998;339,2005-2012[Free Full Text]
  25. Cohen, D., Weinstein, H., Mihm, M., Yankee, R. (1979) Nonfatal graft-versus-host disease occurring after transfusion with leukocytes and platelets obtained from normal donors Blood 53,1053-1057[Abstract/Free Full Text]
  26. Collins, S. J., Gallo, R. C., Gallagher, R. E. (1977) Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture Nature 270,347-349[CrossRef][Medline]
  27. Newburger, P. E., Chovaniec, M. E., Greenberger, J. S., Cohen, H. J. (1979) Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation J. Cell Biol. 82,315-322[Abstract/Free Full Text]
  28. Spellberg, B., Johnston, D., Phan, Q. T., Edwards, J. E., Jr, French, S. W., Ibrahim, A., Filler, S. G. (2003) Parenchymal organ, and not splenic, immunity correlates with host survival during disseminated candidiasis Infect. Immun. 71,5756-5764[Abstract/Free Full Text]
  29. Mashiba, H., Matsunaga, K. (2003) Proliferation inhibition of human cancer cells in combined use of electroporation with attenuated diphtheria toxin Cancer Biother. Radiopharm. 18,803-810[CrossRef][Medline]
  30. Costanzi-Strauss, E., Strauss, B. E., Naviaux, R. K., Haas, M. (1998) Restoration of growth arrest by p16INK4, p21WAF1, pRB, and p53 is dependent on the integrity of the endogenous cell-cycle control pathways in human glioblastoma cell lines Exp. Cell Res. 238,51-62[CrossRef][Medline]
  31. Delissalde, F., Hernandez, M. A., Barron, A., Bermejo, L., Arias, J., Halhali, A., Castro, I. (1998) Vitamin D induces proliferation in rat endometrium cultured cells Rev. Invest. Clin. 50,113-118[Medline]
  32. Tokalov, S. V., Gutzeit, H. O. (2003) The heat shock-induced cell cycle arrest is attenuated by weak electromagnetic fields Cell Prolif. 36,101-111[CrossRef][Medline]
  33. Cormack, B. P., Bertram, G., Egerton, M., Gow, N. A., Falkow, S., Brown, A. J. (1997) Yeast-enhanced green fluorescent protein (yEGFP)a reporter of gene expression in Candida albicans Microbiology 143,303-311[Abstract]
  34. Van t Wout, J. W., Mattie, H., van Furth, R. (1989) Comparison of the efficacies of amphotericin B, fluconazole, and itraconazole against a systemic Candida albicans infection in normal and neutropenic mice Antimicrob. Agents Chemother. 33,147-151[Abstract/Free Full Text]
  35. Chen, L., Zeng, Y., Yang, H., Lee, T. D., French, S. W., Corrales, F. J., Garcia-Trevijano, E. R., Avila, M. A., Mato, J. M., Lu, S. C. (2004) Impaired liver regeneration in mice lacking methionine adenosyltransferase 1A FASEB J. 18,914-916[Abstract/Free Full Text]
  36. Rhyne, A. L., Steel, R. G. (1967) A multiple comparisons sign test: all pairs of treatments Biometrics 23,539-549[CrossRef][Medline]
  37. Dipietrantonio, A., Hsieh, T. C., Wu, J. M. (1996) Differential effects of retinoic acid (RA) and N-(4-hydroxyphenyl) retinamide (4-HPR) on cell growth, induction of differentiation, and changes in p34cdc2, Bcl-2, and actin expression in the human promyelocytic HL-60 leukemic cells Biochem. Biophys. Res. Commun. 224,837-842[CrossRef][Medline]
  38. Matzner, Y., Gavison, R., Rachmilewitz, E. A., Fibach, E. (1987) Expression of granulocytic functions by leukemic promyelocytic HL-60 cells: differential induction by dimethylsulfoxide and retinoic acid Cell Differ. 21,261-269[CrossRef][Medline]
  39. Brieland, J., Essig, D., Jackson, C., Frank, D., Loebenberg, D., Menzel, F., Arnold, B., DiDomenico, B., Hare, R. (2001) Comparison of pathogenesis and host immune responses to Candida glabrata and Candida albicans in systemically infected immunocompetent mice Infect. Immun. 69,5046-5055[Abstract/Free Full Text]
  40. Ibrahim, A. S., Sheppard, D. C., Fu, Y., Edwards, J. E., Jr (2002) Expression analysis of the agglutinin-like-sequence-1 of C. albicans 12th Meeting of the International Immunocompromised Host Society Bergen Norway.
  41. Waldorf, A. R., Halde, C., Vedros, N. A. (1982) Murine model of pulmonary mucormycosis in cortisone-treated mice Sabouraudia 20,217-224[Medline]
  42. Spellberg, B. J., Johnston, D., Phan, Q. T., Edwards, J. E., Jr, French, S. W., Ibrahim, A., Filler, S. G. (2002) Immune response to hematogenously disseminated candidiasis in mice 6th American Society for Microbiology Conference on Candida and Candidiasis Tampa, FL.
  43. Ibrahim, A. S., Bowman, J. C., Avanessian, V., Brown, K., Spellberg, B., Edwards, J. J., Douglas, C. M. (2005) Caspofungin inhibits Rhizopus oryzae 1,3-ß-D glucan synthase, lowers burden in brain by quantitative PCR, and improves survival at a low but not a high dose during murine disseminated zygomycosis Antimicrob. Agents Chemother. 49,721-727[Abstract/Free Full Text]
  44. Spellberg, B., Edwards, J. E., Jr (2001) Type 1/type 2 immunity in infectious diseases Clin. Infect. Dis. 32,76-102[CrossRef][Medline]
  45. Schwarzenberg, I., Mathe, G., de Grouchy, J., de Nava, C., de Vries, M. J., Amiel, J. L., Cattan, A., Schneider, M., Schlumberger, J. R. (1965) White blood cell transfusions Isr. J. Med. Sci. 1,925-956[Medline]
  46. Levin, R. H., Whang, J., Tjio, J. H., Carbone, P. P., Frei, E. I., Freireich, E. J. (1963) Persistent mitosis of transfused homologous leukocytes in children receiving anti-leukemic therapy Science 142,1305-1307[Abstract/Free Full Text]
  47. Lowenthal, R. M., Grossman, L., Goldman, J. M., Storring, R. A., Buskard, N. A., Park, D. S., Murphy, B. C., Spiers, A. S., Galton, D. A. (1975) Granulocyte transfusions in treatment of infections in patients with acute leukaemia and aplastic anaemia Lancet 1,353-358[Medline]
  48. Tonn, T., Becker, S., Esser, R., Schwabe, D., Seifried, E. (2001) Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92 J. Hematother. Stem Cell Res. 10,535-544[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Eukaryot CellHome page
Y. Fu, G. Luo, B. J. Spellberg, J. E. Edwards Jr., and A. S. Ibrahim
Gene Overexpression/Suppression Analysis of Candidate Virulence Factors of Candida albicans
Eukaryot. Cell, March 1, 2008; 7(3): 483 - 492.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
B. J. Spellberg, M. Collins, V. Avanesian, M. Gomez, J. E. Edwards Jr., C. Cogle, D. Applebaum, Y. Fu, and A. S. Ibrahim
Optimization of a myeloid cell transfusion strategy for infected neutropenic hosts
J. Leukoc. Biol., March 1, 2007; 81(3): 632 - 641.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
W. W. Hope, G. L. Drusano, C. B. Moore, A. Sharp, A. Louie, T. J. Walsh, D. W. Denning, and P. A. Warn
Effect of Neutropenia and Treatment Delay on the Response to Antifungal Agents in Experimental Disseminated Candidiasis
Antimicrob. Agents Chemother., January 1, 2007; 51(1): 285 - 295.
[Abstract] [Full Text] [PDF]

Home page
 J Antimicrob ChemotherHome page
B. Spellberg, J. Schwartz, Y. Fu, V. Avanesian, J. Adler-Moore, J. E. Edwards Jr, and A. S. Ibrahim
Comparison of antifungal treatments for murine fusariosis
J. Antimicrob. Chemother., November 1, 2006; 58(5): 973 - 979.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0205072v1
78/2/338    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Spellberg, B. J.
Right arrow Articles by Ibrahim, A. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Spellberg, B. J.
Right arrow Articles by Ibrahim, A. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS