Apr 8, 1987 - The authors gratefully acknowledge the gift of C neofor- mans (145A) from Dr. Judith Domer, the technical help of. Jacky Albertson, and the ...
American Journal of Pathology, Vol. 128, No. 2, August 1987
Copyright © American Association of Pathologists
Role of Natural Killer Cells in Resistance to Cryptococcus neoformans Infections in Mice MARY F. LIPSCOMB, MD, TERESA ALVARELLOS, MD, GALEN B. TOEWS, MD, ROBERT TOMPKINS, BS, ZOE EVANS, PhD, GLORIA KOO, PhD, and VINAY KUMAR, MD
From the Departments ofPathology, Internal Medicine, and Medical Laboratory Sciences, University of Texas Health Science Center at Dallas, Dallas, Texas, and the Department of Immunology Research, Merck, Sharpe, and Dohme Research
Previous studies have suggested a possible role for natural killer (NK) cells in resistance to some fungal infections, including Cryptococcus neoformans infections. The role of NK cells in early clearance of C neoformans from tissues and in long-term survival was studied in mice following intravenous inoculations of the organism. Mice treated with anti-asialo GM1 antiserum to tempoan increase in rarily reduce NKunits activity demonstrated ofC neoformans in the lung colony-forming (CFU) 24 hours after an intravenous inoculation of the organism. CFU in liver, spleen, kidney, and brain were not different in anti-asialo GM1 antiserum-treated versus control mice. An NK-specific reagent, anti-NK 1.1 monoclonal antibody, was used to deplete mice of NK cells in vivo for at least 14 days without affecting other natural defenses. The number of C neoformans retained in the lungs 24 hours after inoculation ofthe organism
was significantly greater in NK cell-depleted mice than in controls, although CFU in other organs were unaf-
CRYPTOCOCCUS NEOFORMANS causes infections in apparently normal individuals, but more commonly infects those who are immunocompromised.' It is an important fungal opportunist in many patients with the acquired immune deficiency syndrome.2 The mechanism of protection against C neoformans infection in the normal host is incompletely understood, but probably requires intact cell-mediated immunity as an important component.3 Thus, both resistance, as measured by growth of the organism in tissue, and survival are decreased in C neoformans-infected athymic nude mice.4'5 Furthermore, immunity has been passively transferred with T-cellenriched splenocytes.6 There is some evidence that natural killer (NK) cells might also play a role in controlling cryptococcal infections. Mice that are homozygous for the beige mutation and have decreased NK cell function are less resistant to C neoformans infections than their normal heterozygous littermates, as measured by
Laboratories, Rahway, New Jersey
fected. Following the intravenous inoculation ofC neothe survival of anti-NK 1.1-treated mice was formans, not different from control mice. The effect of NK cell activity on resistance to C neoformans was also determined after an intratracheal inoculation of the organism. Mice pretreated with anti-NK 1.1 demonstrated no increases in CFU in the lungs, spleen, or brain as compared with controls. These data indicate that NK cells can play a role in vivo in early resistance against C ifthe organism is delivered via the intraveneoformans nous route. However, NK cells do not play a role in either determining survival after an intravenous inoculation nor in resistance during an infection acquired via the respiratory tract. (Am J Pathol 1987, 128:354361)
both early growth inhibition in lung and spleen7 and in long-term survival.8 Furthermore, the replication of C neoformans in vitro is inhibited by splenic NK cells.9 More recently, it has been noted that mice pretreated with cyclophosphamide demonstrated a markedly decreased ability to clear C neoformans from their spleens, liver, and lungs early in infection.'0 A small, but significant, part of the diminished clearance in cyclophosphamide-treated mice was restored by infusion of normal spleen cells, but not with spleen cells that were treated with asialo GM 1 antiserum to deplete NK cells.10 These studies suggested
Supported by NIH Grants AI 21951, AI 20451, and HL Accepted for publication April 8, 1987. Address reprint requests to Mary F. Lipscomb, MD, University of Texas Health Science Center at Dallas, Department of Pathology, 5323 Harry Hines Blvd., Dallas, TX 29543.
75235-9072.
354
ROLE OF NK CELLS IN MURINE CRYPTOCOCCOSIS
Vol. 128 · No. 2
that NK cells might inhibit replication of C neoformans in early infections and, thus, might contribute to prolonging survival in normal hosts. However, previous studies suffered from the lack of specific reagents to remove NK cells and from the absence of a host system where NK cell function was decreased, but other host defenses were intact. In the current studies, we adapted a very sensitive lung clearance assay for in vivo NK cell activity against tumor cells administered intravenously to study the role of NK cells in resistance against C neoformans. We demonstrated that pretreatment of mice with anti-asialo GM1 antiserum to temporarily reduce NK activity resulted in a decreased clearance of C neoformans from the lungs following the intravenous inoculation of the organism. Because asialo GM1 is present on some murine macrophages,'2,13 NK activity was also ablated with the monoclonal antibody NK 1.1, which selectively recognizes only NK cells." After intravenous inoculation of mice with C neoformans, there was a significant decrease in early lung clearance in anti-NK 1.1 antibody-treated mice. This finding verified the earlier studies demonstrating an effect of NK cells on early resistance. However, survival following intravenous inoculation of the organism was not significantly affected in animals that were NK cell-depleted. Furthermore, when organisms were delivered via the respiratory route, animals pretreated with anti-NK 1.1 failed to demonstrate a decreased resistance as measured by quantitating colony-forming units (CFUs) in the lung, spleen, and brain.
Materials and Methods Animals
C57BL/6 mice (Jackson Laboratories, Bar Harbor ME) were used because their NK cells possess the appropriate allotype recognized by the NK 1.1 monoclonal antibody. Animals were kept in a special pathogen-free facility in the Animal Resources Center and monitored for mycoplasma and Sendai virus infec-
tions. All animals were maintained on a pellet diet with water ad libitum. Both male and female mice were used; but within an experiment, all of the animals were of the same sex.
Organism A highly virulent, heavily encapsulated C neoformans strain ( 145, serotype A) was a gift of Dr. Judith Domer (New Orleans, La).14 Stock cultures were maintained by monthly transfer on Sabouraud dex-
355
agar slants with storage at 4 C. The infecting inoculum was prepared bytransferring the stock organism into sterile both medium composed of 1% neopeptone and 2% glucose and culturing for 72 hours at 35 C in ambient air on a gyratory shaker. Cells were harvested, washed three times, and suspended in nonpyrogenic saline to the desired concentration. The number of cells in the inoculum was initially estimated by hemacytometer counts and the CFUs in each inoculum was determined by plating serial dilutions on Sabouraud dextrose agar. trose
Clearance Assays For the lung clearance assay each animal was inoculated via the tail vein with C neoformans in 0.5 ml of nonpyrogenic saline. At 10 minutes groups of 5 animals were sacrificed, and the CFUs in the lung enumerated. Twenty-four hours later a second group of at least 5 animals was sacrificed and CFUs determined in the lung. In some experiments, CFUs were also enumerated in liver, spleen, kidney, and brain. For experiments evaluating clearance following intratracheal inoculation, animals were anesthetized and a 20-gauge blunt needle was inserted through the mouth into the trachea. For intratracheal (IT) bolus inoculation, PE-10 tubing containing 5 u1 of C neoformans in a phosphate-buffered saline (PBS) slurry was inserted into the needle and advanced 7 mm from the tip of the needle, and the organisms were deposited. This technique deposits the inoculum at the level of the tracheal bifurcation. The majority of the organisms are recovered in the left lung 10 minutes after deposition. Groups of four animals were immediately sacrificed for assessment of the number of CFU deposited in the lung by removing the right and left lungs with the attached mainstem bronchi.
Quantitative Culture of C neoformans in Tissue Organs were placed in 2 ml of sterile saline and homogenized either in a Vertis 45 tissue homogenizer (Vertis Company, Gardiner, NY) and then ground in a Ten Broeck tissue grinder (Corning Glass Works, Corning, NY), or the fluid and organs were placed in sterile plastic bags and blended with a Stomacher-80 laboratory blender (Tekmar, Cincinnati, OH) for 3 minutes. The Stomacher only partially disrupted lung tissue. Therefore, lung was also placed in the tissue grinder. Serial 10-fold dilutions of each homogenate were plated in duplicate and incubated at 34 C. CFUs were enumerated at 72 hours.
356
LIPSCOMB ET AL
AJP · August 1987
Antisera The monoclonal antibody PK136 (IgG2b) recognizing the NK 1.1 antigen has been described recently.'5 It was prepared as an ascites fluid containing 2 mg antibody/mi. Control ascites fluid from the mouse inoculated with the fusion partner cell line, Sp2/10, was used in control animals. After the initial fusion, the Sp2/10 line became a secretor and secretes an irrelevant IgG2b monoclonal antibody. Antiasialo GM1 antiserum was purchased from Wako Chemicals (Dallas, Tex). Normal rabbit serum was used as a control. NK Cell Assay Preparation of effector lymphoid cells from spleen and lung has been described.16 Briefly, both spleen and lung were minced. Lung was collagenase-treated and tapped through a stainless steel screen. Mononuclear cells were passed through a nylon wool column, counted, and used as effector cells in the YAC-1 killing assay. YAC-1 Moloney virus-induced T lymphoma cells (H-2a) were previously obtained from the late Dr. G. Cudkowicz (State University of New York, Buffalo, NY). Cells were tested for NK activity with a 4-hour chromium release assay employing labeled YAC-1 as target cells.1' Briefly, 5 X 103 51Cr-labeled targets in 0.1 ml were plated with a varying number of effector cells in triplicate in 96-well round-bottom plates in a total volume of 0.2 ml. After a 4-hour incubation at 37 C in 5% CO2, the plates were spun down for 5 minutes at 60g and 0.1 ml of the supernatant was harvested and counted in a Gamma Trac 1191 Gamma Counter (TM Analytic, Inc., Elk Grove Village, Ill). Data were calculated as: Percent specific cytotoxicity=
experimental cpm
spontaneous cpm maximum cpm spontaneous cpm X 100 Spontaneous release was determined from wells that contained labeled targets alone, whereas maximum release was determined from wells of labeled -
targets which had Zapoglobin (Coulter Diagnostics
Inc., Hialeah, Fla).
Antibody Pretreatment Groups of animals were pretreated with 20 #1
anti-asialo GM 1 antiserum or control rabbit immunoglobulin via an intravenous inoculation. In other
experiments, animals were inoculated intraperitoneally with 30 liters of ascites fluid containing the monoclonal NK 1.1 antibody (60 ug antibody/animal) or control ascites fluid.
Statistics The Student t test for unpaired observations was used for comparison of differences in CFUs between organs of treated and control animals and in mean survival times.
Results The Role of the Lung in the Initial Clearance of an Intravenous Inoculum of C neoformans Previous studies have demonstrated that if YAC-1 lymphoma cells that are sensitive to lysis by NK cells in vitro are inoculated intravenously, they lodge initially in the lungs and are rapidly cleared (within 4 hours) by NK cells.17 Furthermore, in the mouse melanoma model, effective lung clearance correlates with decreased growth of tumor colonies in the lung.I8 Because NK cells have been proposed as important effector cells in the prevention of the growth of C neoformans in vitro and in vivo, we determined whether C neoformans inoculated intravenously were initially trapped in the lung and subsequently rapidly cleared. The 10-minute localization in lung, liver, spleen, kidney, and brain of C neoformans was determined in two experiments. CFUs were also enumerated at 4 and 24 hours. As shown in a representative experiment (Table 1), the majority of C neoformans recovered at 10 minutes after inoculation was in the lung. There was a marked reduction in lung CFUs at 4
Table 1 -Recovery of C neoformans After Intravenous Inoculation*
CFU ± SEMa
Organ cultured Lung Liver
10 minutes 32,000 ± 700 220 ± 20± 160± 38±
Spleen
Kidney
Brain
Total CFU/5 organs
50 10 14 5
32,500 ± 80
*Inoculum 37,000 CFUs, n = 5, at each time point.
4 hours 4200 ± 108 1500 ±200 350± 36 740 62 170± 29
7,000 ± 1,500
24 hours 3300 ± 660 200 ±200 340 ±320 1400 ±120 340± 68 7,900 ± 800
Vol. 128
·
ROLE OF NK CELLS IN MURINE CRYPTOCOCCOSIS
No. 2
357
Table 2-Effect of Anti-Asialo GM1 Antiserum Pretreatment of 24-Hour Clearance of C neoformans After Intravenous Inoculation*
CFU X 10-2 (mean ± SEM)
Experiment
Organ
Anti-asialo GM1
Normal serum
1 2 3
Lung Lung Lung Brain
12.0 ± 0.6t 11.7 + 0.9t 13.3 + 1.9t 33.0 ± 2.3 6.2 ± 0.3 6.6 ± 0.8 0.7 ± 0.1
1.0 0.9 8.4 ± 0.9 6.8 ± 1.1 27.0 ± 3.6 5.5 ± 0.5 6.4 ± 0.5 0.9 ±
