Eur. J. Immunol. 1992. 22: 1447-1454

Helen L. Collins and Gregory J. Bancroft Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London

Cytokine enhancement of complement-dependent phagocytosis

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Cytokine enhancement of complement-dependent phagocytosis by macrophages: synergy of tumor necrosis factor-a and granulocyte-macrophage colony-stimulating factor for phagocytosis of Cryptococcus neoformans* We have examined the regulation of complement dependent phagocytosis by macrophage-activating cytokines. Tumor necrosis factor (TNF)-a and granulocyte-macrophage colony-stimulating factor (GM-CSF), but not interferon-y, interleukin-4 or macrophage-CSF, stimulated ingestion of the encapsulated fungal pathogen Cryptococcus neoformans by resident peritoneal macrophages in vitro. This was dependent upon opsonization of the yeasts with complement, 72 h of incubation with the cytokines for maximum effect, and the obligate involvement of the macrophage CR3 receptor. TNF-a and GM-CSF synergized at low concentrations, resulting in dramatic up-regulation of phagocytosis when compared to either cytokine alone. Supernatants from C. neoformans-specific T cells also increased macrophage phagocytic efficiency. Finally, the administration of neutralizing mAb specific for TNF-a and GM-CSF increased mortality in C. neoformans-infected mice, and induced the rapid progression of disease with involvement of the brain and meninges. We conclude that TNF-a and GM-CSF are potent regulators of complement-dependent phagocytosis by murine macrophages. Macrophage activation with these two cytokines can completely overcome the anti-phagocytic properties of the virulent yeasts. Our results, therefore, implicate TNF-a and GM-CSF as important mediators of resistance to encapsulated pathogens such as C. neoformans where ingestion of the organism is a critical process in host resistance.

1 Introduction Ingestion of pathogens by phagocytic cells is a central process in host defense against infection. Mononuclear phagocytes express several surface receptors that mediate phagocytosis of invading pathogens including Fc receptors and the mannose-fucose receptor. However, of particular importance are the complement receptors CR1 and CR3 which primarily bind the C3 fragments C3b and iC3b, respectively and mediate ingestion of a variety of organisms including Leishmania [ 11, Streptococcus pneumoniae [2] and Candida albicans [3].

In this report we have investigated the regulation of complement-dependent phagocytosis by murine macrophages utilising the pathogenic yeast Cryptococcus neoformans which is a significant fungal pathogen in immunosuppressed individuals. Infection with C. neoformans is an increasingly important problem in AIDS patients and often results in a lethal meningitis [4]. In common with other

[I 102771 ~~

*

This work was supported by an MRC PhD studentship to HLC and a Wellcome Trust University Award to GJB.

Correspondence: Georgy J. Bancroft, Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London WClE, GB Abbreviation:

CN: Cryptococcus neoforman.7

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992

meningeal and pulmonary pat hogens such as Pneumococcus, Neisseria meningitidis and Haemophilus influenzae, synthesis of an anti-phagocytic polysaccharide capsule is the major determinant of virulence for C. neoformans [5, 61. Encapsulated yeasts are not ingested by phagocytic cells unless opsonized with either specific antibody [7] or serum [S]. The opsonic activity of normal human serum is abolished by heat inactivation or the presence of EDTA and occurs via activation of the alternate complement pathway [9, 101. C3 fragments, predominantly in the form of iC3b bind to the outer surface of the polysaccharide capsule [9], facilitating interaction with complement receptors on the surface of phagocytic cells. Furthermore, administration of cobra venom factor to guinea pigs increases susceptibility to C. neoformans, confirming the importance of complement-dependent events in resistance to infection in vivo [lO].Thus,with its absolute requirement for opsonization, virulent C. neoformans is an ideal model to study the regulation of complement receptor-mediated phagocytosis. Complement receptor-mediated phagocytosis is a highly regulated event, although the precise mechanism of this regulation is unclear. While Fc and mannose receptors are constitutively competent for phagocytosis of foreign particles, complement-mediated ingestion by macrophages is inefficient without an external activating stimulus [ l l ] . Interaction with components of the extracellular matrix such as fibronectin and laminin (reviewed in [12]), or pharmacological stimuli such as phorbol esters [13] enhance complement receptor-mediated phagocytosis in human monocytes. While cytokines such as IFN-y, IL-4, 0014-2980192/0606-1447$3.50+ ,2510

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H. L. Collins and G. J. Bancroft

granulocyte-macrophage (GM)-CSF and TNF are known to activate other macrophage functions such as expression of MHC antigens and microbicidal or tumoricidal activity, their effects on complement-dependent phagocytosis have not been extensively examined. Griffin et al. described activation of murine peritoneal macrophages by a T cellderived lymphokine [ 141, although the cytokine responsible and the mechanism of action were not defined. More recently, macrophage (M)-CSF and IL-4 were found to increase ingestion of complement-opsonized erythrocytes in a process involving autostimulation of the macrophages by IFN-(3 [15]. In this report, we have examined a panel of established macrophage-activating cytokines for their effects on complement-dependent phagocytosis. Incubation of resident peritoneal macrophages with either TNF-a and GM-CSF, or supernatants from Tcells specific for C. neoformans enhanced phagocytosis of complementopsonized yeasts. These cytokines were also shown to be critical in the host response to infection with C. neoformans in vivo. These results suggest that complement-dependent phagocytosis is regulated by a distinct subset of macrophage-activating cytokines which are essential for resistance to this important fungal pathogen.

2 Materials and methods 2.1 Cryptococcus neoformans Cryptococcus neoformans var. neoformans (C. neoformans) was used throughout these experiments. An encapsulated, clinical isolate strain B3501 was stored at 4°C in water culture and used in phagocytosis assays and in vivo infections. Opsonization was achieved by incubation with normal mouse serum or culture medium for 1h at 37"C, followed by three washes in assay medium to remove excess serum. An acapsular mutant strain, B4131, derived from B3501 was heat killed by incubation at 80°C for 1 h, followed by three washes with pyrogen-free saline, and stored at 4°C. Both strains of the organism were kindly provided by Dr. J. Kwon-Chung, NIH, Bethesda, MD.

2.2 Mice and cytokines Specific pathogen-free CBA/CA female mice at 8-12 weeks of age were obtained from the National Institute for Medical Research, Mill Hill, London and housed at the London School of Hygiene and Tropical Medicine. Purified murine IL-4 (sp. act. 1 x lo7 U/mg) and GM-CSF were kind gifts from Dr. A . O'Garra, DNAX Research Institute, Palo Alto, CA. Purified recombinant murine TNF-a (sp. act. 1.2 x lo7 U/mg) and IFN-y (sp. act. 1 x lo7 U/mg) were kindly provided by Dr. G. Adolf, Boehringer Ingelheim, Austria. Purified recombinant human M-CSF (sp. activity4.3 X lo7U/mg) [16,17] was kindly provided by Dr. l? Ralph, Cetus Corporation, Emeryville, CA.

2.3 Preparation of C. neoformans-specific T cell supernatants Cell supernatants were harvested from lymph node cells proliferating in response to C. neoformans-pulsed macro-

phage antigen-presenting cells as previously described [HI. In summary, macrophage monolayers prepared from peritoneal exudate cells activated in vivo with 200 pg concanavalin A (Sigma Chemical Co., St. Louis, MO), were allowed to ingest heat-killed acapsular C. neoformans for 2 h, and fixed in 2% paraformaldehyde. Popliteal lymph nodes were harvested from mice injected 7 days previously with 5 x lo7 heat-killed acapsular organisms in incomplete Freund's adjuvant. A single-cell suspension was prepared, the cells were diluted to a final concentration of 5 x 106/ml and added to the macrophages in 100-pl volumes. Cells were incubated at 37 "C, supernatants harvested at 48 h and stored at - 20°C. TNF was measured by ELISA as previously described [ 191 and cytokine titers were determined from a standard curve generated in each assay using rTNF-a.

2.4 Antibodies Neutralizing hamster mAb TN319.12 specific for mouse TNF-a and TNF-(3[19] and L2, an isotype-matched control antibody were kind gifts from Prof. R. D. Schreiber, Washington University, St. Louis, MO. A mAb, MP122E9.11, specific for murine GM-CSF [20] and an isotypematched control antibody, GL117.41, against P-galactosidase were kindly provided by Dr. J. Abrams, DNAX Research Institute, Palo Alto, CA. 5C6 mAb specific for the murine CR3 receptor (CDllb) [21] was a kind gift from Professor S. Gordon, University of Oxford, GB. MU70 (anti-CDllb) and the isotype-matched control antibody llB11 (anti-IL-4) were kindly provided by Dr. F? Kaye, London School of Hygiene and Tropical Medicine, GB. mAb were used as affinity-purified intact immunoglobulins.

2.5 Quantitation of ingestion of C. neoformans by peritoneal macrophages Resident peritoneal exudate cells were harvested by lavage with 10ml of RPMI 1640 supplemented with 1% FCS, 100 IU/ml penicillin, 100 pg/ml streptomycin and 10 mM Hepes buffer (referred to as R1; medium and supplements from Gibco, Paisly, Scotland). Cells were washed once and diluted to a final concentration of 2 x 106/mlin medium as in R1 but containing 10% FCS (R10). Cells were plated in 300-p1 volumes into 8-chamber glass Labtek slides (Gibco), adhered for 2 h at 37 "Cand washed to remove nonadherent cells. Cytokines,T cell supernatants or medium alone were added at appropriate concentrations in 300 pl volumes and cells incubated at 37°C. After 72 h, cells were washed to remove cytokines and 3 x 106 complement-opsonized C. neoformans added in 300-pl aliquots per well. Further incubation for 2 h at 37 "C allowed binding and ingestion to occur. After washing four times to remove unbound organisms, cells were fixed in 100% methanol for 10 min at room temperature to permeabilize the cell membranes. Following washing of the cells in PBS 5% donor calf serum (PBS/DCS; Seralab, Crawley, Down, GB), rabbit anti-C. neoformans capsule antibody (Alpha Laboratories, Hampshire, GB) diluted 1: 500 in PBS/DCS was added to each well. Cells were washed three times in PBS and goat anti-rabbit Ig-TRITC (Nordic Immunologicals, Maidenhead, GB) diluted 1 : 4 0 in PBS/DCS was added. All

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Cytokine enhancement of complement-dependent phagocytosis

stainings were done in 50 yl/well at 4 "C for 1 h. After final washing in PBS,cells were mounted under coverslips and sealed. Organisms were visualized by fluorescence, and ingestion was expressed as C. neoformans (CN) bound or ingested per 100 macrophages.To determine the number of organisms ingested vs. those bound to the cell surface, incubations with rabbit anti-C. neoformans capsule antibody followed by goat anti-rabbit Ig-FITC (Nordic) were performed prior to methanol fixation, to label bound but extracellular organisms. Following methanol treatment, the first-layer staining was repeated, followed by the second-layer staining with goat anti-rabbit Ig-TRITC. The number of organisms ingested was calculated by subtracting the number of yeasts stained green (bound) from the total number of organisms stained red (total boundhgested). 2.6 Measurement of CR3 receptor number CR3 receptor number was determined by solid-phase immunoassay as previously described [22]. All procedures were performed at 4 "C. Peritoneal macrophage monolayers were activated with cytokines for 3 days as described, incubated with 100 yl of normal rabbit serum for 10 min and 300 p1 of biotinylated 5C6 mAb (40 pg/ml) or medium alone added for 30 min. The monolayers were washed, 50000 cpm lZ5I-labeledstreptavidin added per well for 1 h, cells were removed from the chambers with 4 M NaOH and radioactivity was measured. 2.7 Cytokine depletion and infection with C. neoformans in vivo

TN319.12 (anti-TNF) and the isotype-matched control antibody L2 were injected i.p. weekly at a concentration of 300 pg/mouse in pyrogen-free saline [19]. MP1-22E9.11 (anti-GM-CSF) and the isotype-matched control antibody GL117.41 were injected i.p. weekly at a concentration of 500 yglmouse. Mice were injected with antibody at day-1, and the following day infected with 3 X loh B3501 C. neoformans i.p. Times to death of the animals were noted and tissues harvested for histological examination where appropriate. Determination of fungal loads was achieved by grinding tissues in PBS 0.01% Triton X-100 and plating onto Sabaraud's dextrose agar. Plates were incubated at 37 "C, colony-forming units counted after 48 h and expressed as loglo CFU/g tissue.

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3 Results

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inactivation at 56 "C. Thus under these conditions, opsonization of C. neoformans is complement dependent. Twocolor fluorescence studies demonstrated that in all experiments greater than 90% of the yeasts were intracellular, regardless of the state of activation of the macrophage.

To examine the effects of macrophage activation on phagocytic activity, resident peritoneal cells were incubated with recombinant cytokines in vitro and ingestion of serum opsonized C. neoformans determined 3 days later. TNF-a or GM-CSF increased the efficiency of phagocytosis, resulting in a 2-3-fold increase in the number of opsonized yeasts bound or ingested in comparison to unstimulated macrophages (Fig. 1). Again, two-color fluorescence studies confirmed that this was due to increased ingestion rather than simply binding to the cell surface. In contrast, 1-300 U/ml of IFN-y or IL-4 did not increase ingestion above background levels, despite inducing morphological changes characteristic of macrophage activation. M-CSF, did not enhance ingestion unless used at 1000 U/ml, and even at such concentrations, the resulting increase in phagocytosis was less than that observed with tenfold less TNF-a or GM-CSF (data not shown). Titration of TNF-a or GM-CSF revealed similar dose responses for both cytokines with minimal effects seen at 10 U/ml (Fig. 2) and maximum ingestion with 100-300 U/ml. A striking observation was the potent dose-dependent synergy of these two cytokines for activation of phagocytic activity. Addition of TNF-a or GM-CSF alone at < 10 U/ml had no effect, but in combination resulted in a 4-5-fold increase in ingestion (Fig. 3). The addition of TNF-a plus GM-CSF consistently induced the highest level of complement dependent phagocytosis we have observed ( n = 6 experiments), reducing the effective concentration of cytokines by 30-fold and allowing significant activation with 2 0 . 3 U/ml of each cytokine. In addition to increasing the average number of organisms

TNF

IFNy

IL-4

3.1 TNF-a and GM-CSF enhance ingestion of complement-opsonized C. neoformans GM-CSF

Our initial experiments characterized the phagocytosis of C. neoformans by resident peritoneal macrophages in vitro. Encapsulated C. neoformans was poorly ingested due to the anti-phagocytic action of the capsular polysaccharide (12 ? 1 CN boundhngested per 100 macrophages),whereas prior incubation of yeasts with normal mouse serum increased phagocytosis (167 k 7 boundhngested per 100 macrophages). Opsonization was also observed when organisms were incubated in serum harvested from Tand B lymphocyte-deficient scid mice, but abolished by heat

CN PER 100 MACROPHAGES

Figure 1. Effect of cytokines on ingestion of C. rieoformans by peritoneal macrophages. Peritoneal macrophages were incubated with 100U/ml of various cytokines, and ingestion of serumopsonized encapsulated C. neoforrnans was assayed 3 days later. Results are expressed as mean k SD C. neoformans/100 macrophages.

Eur. J. Immunol. 1992. 22: 1447-1454

H. L. Collins and G. J. Bancroft

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u . . ...... 0

1

.

. ......, . . ......, . . ..A 10

MP1-22E9.11 specific for the respective cytokines (medium alone: 72 k 20; TNF-a 100U/ml: 272 f 30; TNF-a + 10 pg/ml TN319.12: 115 f 8; GM-CSF 100 U/ml: 282 f 11; GM-CSF + 10 pg/ml MP1-22E9.11: 84 f 5 - CN boundhngested per 100 macrophages). Isotype-matched control antibodies, L2 and GL117.41 had no effect on cytokinemediated ingestion. Furthermore, activation of macrophages with TNF-a in the presence of anti-GM-CSF mAb, and vice versa, had no effect on the augmentation of phagocytic activity (data not shown). Therefore, the upregulation of phagocytosis observed with individual cytokines appears to be the result of a direct action on the macrophage, rather than via the induction of one cytokine by the other.

Moo

100

CYTOKINE CONCENTRATION U/ML

Figure2. Titration of TNF-a and GM-CSF for phagocytosis of C neoformans by macrophages.TNF-a (0)and GM-CSF (0)were added at the indicated concentrations to resident macrophages and ingestion of serum-opsonized C.neoformans was assayed 3 days later. Results are expressed as mean f SD C. neoformansl100 macrophages.

ingested per macrophage, activation with these cytokines also increased the percentage of macrophages capable of ingestion (% macrophages ingesting CN: medium 46 f 4; TNF-a 78 f 4: GM-CSF 84 f 3;TNF-a GM-CSF 96 f 5 ) . Therefore, the activation of macrophages with a combination of these two cytokines completely abolished the anti-phagocytic effect of the polysaccharide capsule, now resulting in equivalent ingestion of opsonized, encapsulated as acapsular yeasts (626 f 20 vs. 569 f 26 yeasts boundhgested per 100 macrophages, respectively).

+

Macrophage activation by TNF-a or GM-CSF was abolished by addition of neutralizing mAb TN319.12 and

3.2 Cytokine-enhanced ingestion requires complement and involves the CR3 receptor

To confirm that phagocytosis induced by TNF-a and GM-CSF was complement dependent, C. neoformans were opsonized in serial dilutions of normal mouse serum prior to incubation with the macrophage monolayer. In the absence of complement, resident macrophages were unable to ingest significant numbers of yeasts, although this was increased in the presence of serum (Fig. 4). The requirement for binding of complement components to the yeast remained absolute, regardless of the state of macrophage activation. Thus stimulation of macrophages with 10 U/ml of TNF-a plus GM-CSF, enhanced ingestion of opsonized yeasts, but no ingestion was observed with unopsonized C. neoformans (749 f 34 vs. 23 f 4 CN boundhngested per 100 macrophages, respectively). However, prior macrophage activation with either TNF-a or GM-CSFalone, or in combination reduced the amount of serum required for effective opsonization when compared to resident macrophages.

TNFKiY-CSF

GM-CSF

B B

I MEDIUM

% SERUM OPSONUATION

CN PER 100 WCROPHAGES

Figure3. Synergy of TNF-a and GM-CSF on phagocytosis of C. neoformans by macrophages. GM-CSF ( 1 Ulml), TNF-a (10 Ulml), or a combination of the two cytokines were incubated with macrophages and ingestion of serum-opsonized C neoformans was assayed 3 days later. Results are expressed as mean f SD C. neoformansl100 macrophages.

Figure 4. Effect of serum opsonization on ingestion of C. neoformans by cytokine-activated macrophages. Macrophages were incubated for 3 days in a combination of TNF-a and GM-CSF at 10 Ulml (A) or medium alone (W). C. neoformans-opsonized with various concentrations of normal mouse serum were added and ingestion was assayed. Results are expressed as mean f SD C. neoformansl100 macrophages.

Eur. J. Immunol. 1992. 22: 1447-1454

0 1 0

.

.

,

.

.

20

I

Cytokine enhancement of complement-dependent phagocytosis

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. . , . . I

40

80

80

TIME (HOURS)

Figure 5. Kinetics of cytokine activation for macrophage ingestion of C. neoformans. Macrophages were incubated for the approGM-CSF 100 Ulml priate length of time in TNF-a 100 Ulml )(,. (0),10 Ulml each of TNF-a plus GM-CSF (A) or medium (W), and ingestion of serum-opsonized C. neoformans was assayed 3 days later. Results are expressed as mean L SD C. neoformans/100 macrophages.

Previous reports have indicated that iC3b is the predominant complement fragment bound to the surface of serum opsonized C. neoformans [9]. We, therefore, determined whether ingestion involved the expression of macrophage CR3 receptors. Addition of 5C6, a mAb specific for mouse CR3 [21] reduced the phagocytosis of complement-opsonized yeasts to background levels (TNF-a 300 U/ml: 301 +. 8 vs. TNF-a + 10 pg/ml5C6 : 44 t- 7 CN boundhngested per 100 macrophages). Similar results were obtained using another mAb against the CR3 receptor, M1/70, but not with the control antibody 11Bll specific for IL-4 (data not shown). Finally, we investigated the kinetics of cytokine activation of macrophages to ingest C. neoformans. Significant activation by 100 U/ml of either TNF-a or GM-CSF alone did not occur until after 16 h (Fig. 5 ) , reaching a maximum after 72 h of incubation. However, the combination of 10 U/ml TNF-a plus GM-CSF again resulted in extensive ingestion, but activation was now observed within 3 h. Thus, the combination of TNF-a and GM-CSF enhanced both the kinetics and magnitude of complement receptor-mediated phagocytosis, allowing efficient ingestion even under conditions of limiting opsonization. The kinetics of this response argues against the rapid movement of preformed complement receptors to the macrophage surface as the mechanism for the observed increase in phagocytosis. Furthermore, measurement of CR3 receptor number by solid-phase immunoassay confirmed that the cytokine-enhanced phagocytosis observed is not mediated by an increase in receptor expression on the macrophage surface (medium 1991 t- 300; TNF-a 1546 -t 213; GM-CSF 2577 f 258;TNF-a GM-CSF 2523 t- 550; background 444 k 140cpm).

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3.3 Cryptococcus-specificT cell supernatants up-regulate complement-dependent phagocytosis

Resistance to C. neoformans is primarily dependent on cell-mediated immunity and loss of Tcell function, such as

% T-CELL SUPERNATANT ADDED TO MACROPHAGES

Figure 6. Effect of C. neoformans-specific Tcell supernatants on activation of macrophages for phagocytosis. C. neoformansspecific T cells were incubated with macrophage antigen-presenting cells pulsed with 3 x lo7 acapsular yeasts (A) or medium alone (A), and supernatants harvested at 48 h. Supernatants were incubated with resident peritoneal macrophages and ingestion of serum-opsonized C. neoformans was assayed after 3 days. Results are expressed as mean f SD C. neoformansl100 macrophages.

in AIDS, is associated with increased susceptibility to infection. We, therefore, asked whether C. neoformansspecificT cells also secreted cytokines capable of enhancing phagocytosis of the encapsulated yeast. Supernatants harvested from C. neoformans-primed T cells stimulated in vitro with acapsular yeasts were tested for their effects on macrophage phagocytic activity. Incubation of macrophages with these supernatants resulted in a threefold increase in yeast ingestion compared with unstimulated

T-CELL SUPERNATANT + TN319.12

rTNF + 774319.12

1

m 0

100

2w

300

4w

!

m

CN PER 100 MACROPHAGES

Figure 7. Effect of anti-TNF mAb on Tcell supernatant-induced ingestion of C. neoformans by macrophages. T cell supernatants (50% vlv), or TNF-a (300 Ulml) were added to macrophages in the presence of medium alone or 100 pglml of TN319.12, and ingestion of serum-opsonized C. neoformans was assayed after 3 days. Results are expressed as mean ?: SD C. neoformans/100 macrophages.

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H. L. Collins and G. J. Bancroft

macrophages (Fig. 6). Supernatants harvested fromprimed Tcells cultured in the absence of antigen had only marginal effects on ingestion. Furthermore, addition of mAb TN319.12, at 10 pg/ml (a concentration which inhibited the action of rTNF-a), abolished the capacity of the Tcell supernatants to stimulate ingestion (Fig. 7). Similarly, anti-GM-CSF mAb MP1-22E9.11 at 10 pg/ml, also reduced the activity of T cell supernatants to background levels, in contrast to the isotype-matched control antibody, GL117.41 (medium alone: 131 f 15;Tcell supernatant: 322 k 13; Tcell supernatant anti-GM-CSF: 141 f 9; Tcell supernatant GL117.41: 269 f 8 CN boundhgested per 100 macrophages). Active T cell supernatants contained low, but detectable levels of TNF when measured by ELISA (< 15 U/ml), a concentration which alone has minimal effect on macrophage phagocytosis.

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+

anti-GM-CSF group and 5/5 of the control antibody-treated animals. In other experiments, analysis of fungal loads in tissues of TN319.12-treated mice revealed that by day 13, 100% of animals had yeasts present in the brain and meninges (mean loglo CFU = 4.4 f 0.8), compared to none of the control antibody-treated animals (limit of detection of assay 50 CFU). Systemic organs such as liver, spleen and kidney in the TN319.12-treated mice contained significant numbers of organisms although these were not significantly different from the control antibody group. Thus, administration of neutralizing mAb to TNF-a//B and GM-CSF in vivo leads to the rapid progression of experimental cryptococcosis, with increased mortality and meningeal infection characteristic of this disease in the immunocompromised human host.

4 Discussion 3.4 The effect of anti-TNF and anti-GM-CSF mAb on infection with C. neoformans in vivo Infection of mice with encapsulated C. neoformans results in a lethal infection, with characteristic involvement of the brain and meninges. Given the stimulatory effect of TNF-a and GM-CSF on macrophage phagocytosis of opsonized yeasts in vitro, we investigated whether neutralization of these cytokines in vivo altered resistance to infection. Mice were pretreated 1 day prior to infection and weekly thereafter with 300 pg mAb TN319.12 (specific for TNF-a and TNF-f3) and/or 500 pg of MP1-22E9.11 (specific for GM-CSF) or the respective control mAb L2 and GL117.41. TN319.12 has previously been shown to neutralize TNF in vivo, exhibits a serum half-life of 7 days, and confers protection against TNF-mediated endotoxin shock [19], while MP1-22E9.11 has been demonstrated to neutralize the effects of GM-CSF in vitro [20]. Treatment with TN319.12 or MP1-22E9.11 alone enhanced the severity of infection with C. neoformans and reduced the initial time to death of infected animals (Fig. 8). Furthermore, treatment of animals with a Combination of the two antibodies resulted in an acute infection with 0/5 animals surviving at day 5 in the combined anti-TNF/anti-GM-CSF groups as compared with 1/5 in the anti-TNF group, 2/5 in the

In this study we have utilized the pathogenic yeast Cryptococcus neoformans, to investigate the regulation of complement-dependent phagocytosis by murine macrophages. Encapsulated C. neoformans provides an ideal phagocytic target for these experiments by virtue of its obligate requirement for opsonization and the central role of macrophages in resistance to infection [23].Three major observations arose from these experiments. First, the recombinant cytokines TNF-a and GM-CSF synergized in vitro t o activate macrophages for complement-dependent phagocytosis. Second supernatants from T cells reactive against C. neoformans also secreted cytokines including TNF which enhanced ingestion of virulent yeasts. Finally, administration of neutralizing mAb to TNF and GM-CSF increased susceptibility to experimental infection with C. neoformans in vivo suggesting these cytokines also play active roles in host resistance.

We initially compared a panel of known macrophageactivating cytokines for their ability to stimulate resident peritoneal macrophages to ingest opsonized C. neoformans. Incubation with 10-100 U/ml murine rTNF-a or rGM-CSF increased phagocytic activity, consistent with concentrations required for other parameters of macrophage activation including synthesis of MHC class I1 molecules [24] and intracellular killing of Leishmania [25]. However, the combination of TNF-a and GM-CSF resulted 100 in maximal ingestion. The potent synergy observed with these two cytokines allowed significant macrophage activation at 30-fold lower concentrations of either cytokine alone, with consistent enhancement at 2 0.3 U/ml. Furthermore, addition of both TNF and GM-CSF reduced the amount of complement required for effective opsonization of encapsulated yeasts. TNF-a and GM-CSF are known to synergize for growth of myeloid leukemia cells [26], but to our knowledge this is the first evidence of their potent 0 synergy for functional activation of macrophages. Experi0 5 10 15 20 25 30 ments are in progress to assess their combined effects on DAYS POST INFECTION other macrophage activities. Individually, TNF and GMFigure 8. Effect of anti-TNF and anti-GM-CSFmAb on infection CSF are known to increase other receptor-mediated phawith C. neoformans in vivo. Mice were injected i.p. with 300 pg gocytic events. TNF activates granulocytes to internalize TN319.12 (anti-TNF: O ) ,500 pgMP1-22E9.11 (anti-GM-CSF: 0), TN319.12/MP1-22E9.11 (A),control mAb L2/GL117.4 (+) or complement-opsonized Herpes simplex virus [27] while pyrogen-free saline (0)1 day prior to i.p. infection with 3 x lo6 GM-CSF can increase Fc receptor-mediated ingestion [28]. encapsulated C. neoformans and weekly thereafter. Results show However, the latter mechanism did not account for the % survivors in each group ( n = Ygroup) during the course of results presented here, since opsonic activity of nonimmune serum was heat sensitive and serum from T- and infection.

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B cell-deficient scid mice was equally effective as immunocompetent controls in promoting ingestion. In contrast, IFN-y, a potent activator of other macrophage functions, did not enhance phagocytosis of C. neoforrnans, consistent with reports that IFN-y-activated macrophages can inhibit the growth of C. neoformans without altering the ingestion of encapsulated yeasts [29]. IL-4 also had no effect on phagocytic activity despite inducing changes in cell morphology consistent with macrophage activation. Marginal increases in phagocytosis with M-CSF were only observed at concentrations of 2 1000 U/ml. This is in contrast to recent experiments demonstrating that both IL-4 and M-CSF can increase ingestion of either complement- or Ig-opsonized erythrocytes, via the production of IFN-P[15]. This most likely reflects differences in receptor modulation required for ingestion of microorganisms such as C. neoforrnans in comparison to opsonized erythrocytes. Thus, disruption of actin microfilaments blocks binding of H . capsulaturn or opsonized C. neoforrnans to human monocytes but has no effect on binding of opsonized erythrocytes [30, 311. The mechanism by which TNF-a and GM-CSF up-regulate the efficiency of complement-dependent ingestion is not known. Activation of resident macrophages with these cytokines increases both the number of macrophages capable of phagocytosis, and the average number of yeasts ingested per macrophage. Phagocytosis of C. neoformans induced by TNF-a and GM-CSF is still dependent on the presence of complement, as unopsonized organisms were not ingested. Expression of CR3 is critical for this event since addition of anti-CR3 mAb 5C6 or MU70 block ingestion, consistent with the predominant expression of iC3b on the surface of serum opsonized yeasts [9]. However, binding of C. neoforrnans to cultured human macrophages 1301, can be blocked by mAb directed against CR 1, CR3 or CR4, suggesting the involvement of multiple complement receptors. Therefore, although our experiments have demonstrated internalization of the organism in a process that involves the CR3 receptor, this may not be the only receptor-ligand interaction required and regulated by these cytokines. Addition of TNF-a or GM-CSF resulted in maximal ingestion after 72 h and required at least 16 h in culture to induce significant up-regulation. However, the combination of TNF-a plus GM-CSF allowed an increase in phagocytosis to be observed within 3 h. The lack of measurable changes within several minutes o f cytokine addition argues against a role for rapid translocation of preformed complement receptors to the cell surface as described for TNF on human neutrophils 132,331. In our system, increased ingestion did not occur as a result of a quantitative increase in the surface expression of CR3 as determined by radioimmunoassay. In other models activation of complement receptor-mediated phagocytosis has correlated with aggregation of CR3 and/or changes in receptor mobility within the cell membrane 134,351and our future studies will address the contribution of these processes to cytokine-mediated ingestion of C. neoformans. In addition to the biology of macrophage phagocytic receptors, this study also provides new information on mechanisms of immunity to C. neoformans. Natural infection with C. neoformans occurs via inhalation of poorly encapsulated yeasts, some of which then synthesize an

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extensive polysaccharide capsule in the host. Expression of this anti-phagocytic structure is the primary determinant of virulence in vivo and both macrophage and T cell function are essential for resistance. Following the effects of recombinant TNF-a and GM-CSF in vitro, we asked whether soluble products from C. neoformans-specific T cells could also increase phagocytic activity. Supernatants from lymph node T cells primed against acapsular yeasts activated resident macrophages for increased phagocytosis of C. neoformuns. These supernatants contained low but detectable levels of TNF and were neutralized by addition of mAb specific for either TNF or GM-CSF (but not anti-IFN-y or control antibodies), consistent with the action of multiple cytokines for macrophage activation. Thus, in the immunocompetent host, T cell responses initiated against readily phagocytosed acapsular yeasts may activate macrophages for ingestion of virulent organisms following capsule synthesis in vivo. We have recently demonstrated that capsule synthesis by C. neoforrnans prevents specificT cell proliferation to cryptococcal antigens in vitro [18]. This is mediated by the anti-phagocytic action of the capsule on ingestion by antigen-presenting macrophages rather than an inhibitory effect of capsular polysaccharides on intracellular pathways of antigen processing. Thus, increased uptake by antigenpresenting macrophages mediated by cytokines such as TNF-a and GM-CSF should also promote the further development of T cell-mediated immunity. While extracelM a r killing of C. neoformans has been reported in vitro 1361, it is likely that host mechanisms which increase yeast ingestion will also promote more efficient intracellular fungistasis via the L-arginine-dependent pathway [37]. Together, these results suggest that secretion of phagocytosis promoting cytokines may contribute to T cell-dependent resistance to C. neoformans. The importance of these cytokines for resistance in vivo was confirmed in experiments utilizing neutralizing mAb specific for TNF-a or GM-CSF. TN319.12, a mAb specific for TNF-a,B, has previously been used in assessing the role of TNF in resistance to other intracellular pathogens such as Listeria rnonocytogenes [38]. Administration of TN319.12 prior to, and during infection with C. neoformans, resulted in increased mortality in infected mice, with all treated animals demonstrating the presence of yeasts in the brain, in contrast to none of the control animals. Furthermore, the treatment of mice with a combination of TN319.12 and MP1-22E9.11 (specific for anti-GM-CSF) resulted in a uniformly lethal infection within 6 days and in preliminary experiments was associated with an increase in systemic tungal loads. It is important to note that in addition to its effect on phagocytosis seen here, neutralization of TNF in vivo may also impair other macrophage functions such as stimulation of the L-arginine-dependent pathway 1391, known to be important for fungistasis of C. neoforrnans in vitro 1371. However, these results are consistent with the importance of both TNF and GM-CSF in vivo in resistance to virulent C. neoformans. In conclusion, we have demonstrated that a subset of macrophage-activating cytokines, namely TNF-a and GMCSF are potent inducers of macrophage complement receptor-dependent phagocytosis using C. neoforrnans as a model.The synergistic action of these two cytokines in vitro

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H. L. Collins and G . J. Bancroft

suggests that low concentrations of TNF-a and GM-CSF may be sufficient to enhance uptake of encapsulated C. neoformans in vivo. Indeed, in the presence of complement, macrophage activation by TNF-a plus GM-CSF can completely overcome the anti-phagocytic action of the polysaccharide capsule which is the primary determinant of virulence for C. neoformans. We believe that synthesis of phagocytosis-promoting cytokines may be an important host defense against encapsulated pathogens such as C. neoformans where avoidance of ingestion by phagocytic cells is a key strategy for survival. Furthermore, since complement receptors also mediate ingestion of a wide range of other pathogens including Leishmania, Mycobacterium leprae and Legionella, events described here may also contribute to resistance or pathology in other microbial systems. Finally, our results suggest that in addition to IFN-y [40],TNF-a and GM-CSF are essential mediators of resistance to experimental infection with C. neoformans. Understanding the role of these cytokines in immunity may provide a basis for therapeutic application of recombinant cytokines in the increasing number of AIDS patients infected with this organism. Received January 13, 1992

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