Binding of Candida albicans Yeast Cells to Mouse Popliteal Lymph ...

INFECrION AND IMMUNITY, Aug. 1993, p. 3244-3249 0019-9567/93/083244-06$02.00/0 Copyright © 1993, American Society for Microbiology

Vol. 61, No. 8

Binding of Candida albicans Yeast Cells to Mouse Popliteal Lymph Node Tissue Is Mediated by Macrophages YONGMOON HAN,1 NICO vAN ROOIJEN,2 AND JIM E. CUTLER`* Department of Microbiology, Montana State University, Bozeman, Montana 59717,1 and Department of Cell Biology, Vrije Universiteit, Amsterdam, The Netherlands2 Received 24 February 1993/Accepted 13 May 1993

We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene diphosphonate (LCI2MDP). Mice were injected in the hind footpads with the L-Cl2MDP preparation, popliteal lymph nodes were removed 5 days later, and yeast cell adherence was determined by an ex vivo binding assay. As controls, lymph nodes from mice that received footpad injections of either phosphate-buffered saline (PBS) alone or liposome-containing PBS were used. Use of macrophage- and neutrophil-specific monoclonal antibodies in tissue immunostaining showed that the L-Cl2MDP treatment eliminated macrophages but not neutrophils from the medullary and subcapsular sinus areas of the popliteal lymph nodes. A striking reduction of yeast cell adherence occurred with lymph nodes from L-Cl2MDP-treated mice compared with lymph nodes from control animals. The lymph node-yeast cell binding patterns of L-Cl2MDP-treated and control mice were the same regardless of mouse strain, sex, or T-cell competency. Results of India ink experiments, in which India ink was injected into footpads of mice and was rapidly taken up by popliteal lymph node macrophages, showed a strong correlation between yeast adherence and India ink staining of cells. In addition, the interaction of yeast cells with lymph node tissue from normal mice was not significantly affected by the addition of two extracellular matrix proteins, fibronectin and laminin, during the ex vivo adherence assay. These data indicate that medullary and subcapsular sinus lymph node macrophages express an adhesion system similar to that described for mouse splenic marginal zone macrophages.

MATERIALS AND METHODS Organism and culture conditions. C. albicans A9 was used throughout the study and was previously characterized (12). Prior to the ex vivo binding assays, cultures were started from -20°C glycerol stock cultures as described previously (18), and the yeast cells were grown in glucose (2%)-yeast extract (0.3%)-peptone (1%) broth (GYEPB) for 24 h at 37°C on a shaker incubator (180 rpm; New Brunswick Scientific Co., Inc., Edison, N.J.). The stationary-phase culture was serially transferred to 25 ml of fresh GYEPB in a 50-ml Erlenmeyer flask four times at 24-h intervals and incubated under the same conditions as described above. This procedure ensured production of essentially 100% hydrophilic yeast forms (12). Ex vivo adherence assay. Lymph node tissue was obtained from female mice of strains BALB/cByJ, BALB/cByJ nu/nu athymic (nude), and [BALB/cByJ x Crl:CD-1(ICR)BRJF1, 8 to 11 weeks old. All experiments were done on T-cellcompetent animals unless otherwise specified. The binding assay was done as previously described (18, 23), with few modifications. Briefly, popliteal lymph nodes were removed from mice as previously described (19). Unlike animals used for studies of yeast adherence to splenic tissue (18), these mice were not treated with luconyl blue. The lymph nodes were immediately submerged in Tissue-Tek O.C.T. compound (Miles Inc., Elkhart, Ind.), rapidly frozen, and stored at -80°C before further use. Cryosections (10 ,um thick) (Frigocut 2800 N Cryostat; Reichert-Jung, Leica, Inc., Deerfield, Ill.) were cut and collected on glass slides. After the sections were air dried for 20 min, 100 ,ul of a yeast cell suspension (1.5 x 108 cells per ml) in cold Dulbecco modified

Adherence of Candida albicans to host organs and tissues is thought to be a critical event in the pathogenesis of disseminated candidiasis (10). C. albicans expresses surface molecules (adhesins) that mediate attachment to various types of epithelial cells and plastic surfaces (11, 21, 26). Four categories of adhesin molecules have been described (4, 7), and mannoproteins of the fungal cell wall may be the most significant adhesins (4, 5, 7). Recent work from our laboratory strongly suggests that the mannan portion of the mannoproteins accounts for the adherence of C albicans yeast cells to mouse spleen and lymph node tissues (17). Previously, we reported that yeast cells adhere to spleen and lymph node tissues (8, 12) and that mouse splenic marginal zone macrophages, but not red pulp monocytes, are responsible for the specific binding of hydrophilic yeast cells (18). By using an ex vivo adherence assay, which was adapted from studies on lymphocyte homing receptors (3, 16, 28), and by using in vivo experiments, we concluded that the splenic marginal zone macrophages had a unique adhesion system (18). In the present study, the ex vivo adherence assay was used to identify the predominant C. albicansbinding cell in mouse popliteal lymph nodes. We found that macrophages within the medullary and subcapsular sinuses of lymph node tissue appear to have an adhesion system similar to that of splenic marginal zone macrophages.

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Corresponding author. Electronic mail address:

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C. ALBICANS YEAST CELL ADHERENCE TO LYMPH NODE TISSUE

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FIG. 1. Ex vivo binding of C albicans yeast cells to lymph node tissues from mice treated with PBS (A), L-PBS (B), or L-Cl2MDP preparation (C). Cryosections of lymph node tissue from mice that had been injected in the hind footpads with each of the preparations were allowed to interact with 100 pAl of yeast cells (1.5 x le~cells per ml in CDMEM) for 15 min at 4°C. The sections were then fixed in glutaraldehyde, stained with crystal violet, and examined under bright-field microscopy. In panels A and B, yeast cells bound especially to medullary and subcapsular sinuses of lymph nodes (arrows),

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Eagle medium (Sigma Chemical Co., St. Louis, Mo.) containing 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (GIBCO, Grand Island, N.Y.) and 5% fetal bovine serum (Hyclone Laboratories, Inc., Logan, Utah) and adjusted to pH 7.4 (CDMEM) was added to the sections and incubated for 15 min at 4°C. Yeast cell adherence to lymph node tissue is not altered if fetal bovine serum is either heat inactivated (56°C, 30 min) or omitted from the assay. The sections were fixed and washed, and adherent yeast cells were stained with crystal violet as described previously (18, 23). In some experiments, the effect of extracellular matrix (ECM) glycoproteins on C. albicans yeast cell binding to lymph node tissues was tested. Two kinds of ECM proteins, fibronectin and laminin (Sigma), were used in this study. In accordance with the manufacturer's instructions, each original ECM protein was aliquoted and stored at -20°C. The final protein concentration used in the ex vivo binding assay was 100 nM as previously described (1, 22). Each ECM protein was mixed with a yeast cell suspension (1.5 x 108 cells per ml) in CDMEM, incubated in ice for 15 min, and mixed three to four times during the incubation, and each protein-yeast cell suspension was tested in the ex vivo adherence assay as described above. Control yeast cells were treated similarly, but in medium without the ECM proteins. We tested the activity of the fibronectin in vitro as previously described (27), and we tested the activity of laminin in a similar way, by attaching each to a plastic surface (13, 27) and determining whether C. albicans yeast cells would adhere (27). Wells of a 24-well microtiter plate (Nunc Multidish; Irvine Scientific, Santa Ana, Calif.) were coated by adding to each well 200 ,ul of each ECM protein (70 p,g) in Dulbecco's phosphate-buffered saline, pH 7.4 (PBS) (Sigma) and allowing the wells to air dry. Control wells received PBS and were allowed to air dry. After air drying, all wells were washed with PBS, drained, and overlaid with 100 pl of a C albicans yeast cell suspension (1.5 x 108 cells per ml) in CDMEM, and the plate was incubated at 30°C for 30 min on a gyratory shaker-incubator (150 rpm). The attached yeast cells were fixed in glutaraldehyde (1.5%) and stained with crystal violet as previously described for the ex vivo assay (18, 23). Yeast cell binding to the coated well surface was measured under bright-field microscopy at a magnification of x300. The 10 densest fields of yeast cells adhering to each coated well were measured and expressed as the mean of yeast cells per field. Variance was expressed as standard error. Quantification of yeast cell adherence to lymph node tissue sections. Ex vivo binding of C. albicans yeast cells to lymph node sections was measured by computer image analysis as previously described (23), with the following modifications. The Ml program update of MCID (Imaging Research, St. Catharines, Ontario, Canada) was used; each lymph node section was examined with the lOx objective lens of a microscope; and the image was projected through a video camera, displayed on a color monitor, and digitized as before (23). For each tissue section, 30 contiguous fields (20 by 200 ,um) were sampled from the medullary and subcapsular sinus regions. As noted previously (8), these regions consistently showed specific binding of hydrophilic yeast cells to lymph

whereas in panel C, binding of yeast cells to lymph node tissues treated with L-Cl2MDP was essentially eliminated. Bar, 100 pum.

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node tissue. Measurements were taken from 12 tissue sections (4 sections per slide) for each experimental treatment, results were expressed as the mean of adherent yeast cells per field, and variance was expressed as standard error. Elimination of lymph node macrophages. To eliminate macrophages in popliteal lymph nodes, liposome-containing dichloromethylene diphosphonate (L-Cl2MDP) was prepared and used as described previously (9, 30, 31). C12MDP was a kind gift of Boehringer Mannheim GmbH, Mannheim, Germany. Briefly, 50 ,ul of the L-Cl2MDP suspended in PBS was injected into the hind footpads of mice. Five days later, the popliteal lymph nodes were removed for use in the ex vivo binding assay. For control mice, either 50 ,ul of liposome-containing PBS or 50 pl of PBS alone was injected into the footpads in place of L-C12MDP. Immunoperoxidase staining was used to monitor the effect of L-Cl2MDP on the popliteal lymph nodes. Cryosections (5 ,m thick) of lymph nodes were cut, collected on slides (four sections per slide), air dried for 20 min, fixed in cold acetone for 10 min, and air dried again. The fixed sections were overlaid with 100 pl of each of the monoclonal antibodies (MAbs) listed below at a concentration of 50 jig/ml prepared in RPMI 1640 medium (Sigma) containing 10 mM HEPES (GIBCO) plus 10% fetal bovine serum (Hyclone) and adjusted to pH 7.4, and they were then incubated at 21 to 23°C for 30 min. The rat anti-mouse MAbs were MONTS-4 (specific for macrophages in the splenic marginal zone and the subcapsular sinus of peripheral lymph nodes) (14, 32), SK-39 (specific for monocytes in splenic red pulp and in lymph node tissue) (14), and SK-208, which recognizes the same neutrophil-specific antigen as SK-105 (15). All of the rat MAbs were a gift from Mark A. Jutila, Montana State University. The sections were washed by dipping the slides twice in 300 ml of PBS (pH 7.4) and dipping them once in 300 ml of PBS containing 2 to 3 drops of normal rabbit serum (Sigma). One hundred microliters of a 2.5% biotinylated goat anti-rat immunoglobulin (TAGO, Inc., Burlingame, Calif.) in PBS was overlaid onto the sections, and the sections were incubated for 30 min at 21 to 23°C and washed as before. The washed sections were incubated for 20 min with 100 ,ul of streptavidin peroxidase conjugate (TAGO) that was diluted 1:500 in PBS. The sections were washed as before, overlaid with 100 pl of substrate solution, and incubated for 10 min to allow color development. The substrate solution was prepared fresh before each use as follows. Aminoethylcarbazol (Sigma) and N,N-dimethylformamide (Sigma) were mixed at a ratio of 4 mg to 1 ml, respectively, and stored at -20°C. One part of the mixture was then put into 14 parts of acetate buffer solution, and hydrogen peroxide was added to a final concentration of 0.03%. Subsequently, the sections were rinsed in cold tap water, overlaid with GEL/MOUNT (Biomeda Corp., Foster City, Calif.), and covered with a glass coverslip. Control sections were treated in the same manner but without addition of primary MAbs. India ink tagging of lymph node macrophages. To further examine the cell type to which yeast cells bind, 50 pl of India ink (waterproof drawing ink no. 4415; Faber-Castell Corp., Newark, N.J.) diluted 1:4 in PBS was injected into the hind footpads of mice. Fifteen minutes later, popliteal lymph nodes were removed and used in the ex vivo adherence assay as described above. RESULTS Effect of L-Cl2MDP on C. albicans binding to lymph nodes. Lymph nodes removed 5 days after injection of PBS into the

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FIG. 2. Effect of L-C12MDP on ex vivo binding of C albicans yeast cells to lymph node tissues from [BALB/cByJ x Crl:CD1(ICR)BR]F1 (A) and BALB/cByJ (B) female mice. Cryosections of lymph nodes of mice that had been injected in the hind footpads with PBS, L-PBS, or L-C12MDP were allowed to interact with 100 Ill of yeast cells (1.5 x 108 cells per ml in CDMEM) for 15 min at 4°C. The sections were then fixed in glutaraldehyde and stained with crystal violet. The y axis shows the average number of yeast cells that bound per field (bars indicate standard errors), and thex axis shows different treatments of lymph nodes. Yeast cell binding was measured by computer image analysis. Yeast cell binding to lymph node tissue treated with L-C12MDP was reduced by approximately 92% compared with binding to control tissues that had received either L-PBS or PBS alone.

hind footpads of mice and used in the ex vivo assay showed that C. albicans yeast cells bind especially to medullary and subcapsular sinuses of popliteal lymph nodes (Fig. 1A and 2). Lymph nodes from mice that received the L-PBS showed binding which was almost equal to that of lymph nodes from mice treated with the PBS (Fig. 1B and 2). In contrast, when the mice were injected with the L-Cl2MDP, yeast cell binding to lymph node tissues was significantly reduced (Fig. 1C and 2). The same results were obtained with different mouse strains (Fig. 2), and male and female mice of each strain behaved similarly (data not shown). In experiments done on congenitally thymic-deficient (nude) mice, the number of yeast cells per field averaged 51 + 1.9 (mean + standard error), whereas in nude mice treated with L-Cl2MDP, binding was reduced to 1.7 ± 0.17 yeast cells per field. Effect of L-Cl2MDP on macrophages in lymph node tissue. To determine whether the significant reduction of C albicans yeast cell binding to lymph nodes from the L-Cl2MDPtreated mice correlated with macrophage elimination, immunoperoxidase staining was done. When tissue sections from the PBS-treated mice were immunoperoxidase stained with the macrophage-specific MAb, MONTS-4, and a monocytespecific MAb, SK-39, both cell types in the lymph nodes were found mostly distributed throughout the medullary and subcapsular sinus regions (Fig. 3A and B). In tissue sections of lymph nodes from mice that had been treated with L-Cl2MDP, the monocyte/macrophage cell population was not detectable (Fig. 3C and D). Sections stained with SK208, a neutrophil-specific marker, showed the same cell concentration and tissue distribution patterns, regardless of whether treatment was with PBS or L-C12MDP (Fig. 3E and F). No staining occurred in control tissue sections, in which primary MAb treatments were omitted (data not shown). Correlation of yeast cell binding with India ink-tagged macrophages. Under bright-field microscopy at low power (40x), lymph nodes taken from animals that received India ink showed black staining mostly in the medullary and


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FIG. 3. Effect of L-Cl2MDP on macrophages in lymph node tissue from [BALB/cByJ x Cr1:CD-1(ICR)BR]Fl mice. Five days after injection of either L-Cl2MDP or PBS into the hind footpads of the mice, lymph nodes were removed from the mice for immunoperoxidase staining. When tissue sections of the PBS-treated mice were stained with MONTS-4 (A) or SK-39 (B), resident macrophages were found in the medullary and subcapsular sinuses (arrows). In contrast, when sections treated with L-Cl2MDP were stained with MONTS-4 (C) or SK-39 (D), macrophages were not detected in the medullary and subcapsular sinuses. In SK-208-stained sections of tissues obtained from mice treated with either PBS (E) or L-Cl2MDP (F), resident neutrophils (arrows) were found in both and in essentially identical concentrations. Bar, 100 pm.

subcapsular sinus regions of the lymph node tissue in a pattern very similar to that observed with immunoperoxidase staining for macrophage distribution in the lymph node (Fig. 4A). The pattern of ink staining correlated well with yeast cell distribution (Fig. 4B and C), suggesting that the

cells that ingest ink particles also bind C. albicans yeast cells. Effect of ECM proteins on C. albicans binding to lymph node tissue. Coating of plastic wells with the fibronectin and laminin preparations caused yeast cells to adhere. The

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numbers of yeast cells adhering to coated wells were 157 ± 1.4 and 178 14.1 cells per field for fibronectin and laminin, respectively. Control wells treated with PBS alone resulted in 10.4 3.6 yeast cells per field. However, preparations of fibronectin and laminin did not cause reduced binding of yeast cells in the ex vivo assay. The numbers of yeast cells that bound to the medullary and average

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INFECT. IMMUN.

subcapsular sinuses of lymph node sections averaged 25 ± 2.0 and 24 ± 2.4 per field in the presence of fibronectin and laminin, respectively. Positive binding controls, in which yeast cells were not exposed to the ECM proteins, showed 27 ± 3.7 yeast cells per field. DISCUSSION We previously demonstrated that C. albicans hydrophilic yeast cells bind to mouse splenic and lymph node tissues rich in macrophages (8, 12). More recently, yeast cell adherence to the marginal zone of the spleen was found to be mediated by macrophages but not by red pulp macrophages or by phagocytic cells in the thymus (18). Furthermore, the adhesion system involved in yeast cell binding was not due to other known systems such as those involving integrins and extracellular matrix proteins (18). In the present study, we confirmed that hydrophilic C. albicans yeast cells preferentially bind to the medullary and subcapsular sinuses of lymph node as reported before (2, 8, 12). In consideration of the possibility that the interaction between the yeast cells and lymph node tissue may be mediated by macrophages, we used a toxic liposome preparation (L-Cl2MDP) to eliminate lymph node macrophages (9, 31) and determined this effect on yeast cell binding to the tissues. The L-Cl2MDP treatment of mice caused almost total elimination of yeast cell binding to the lymph node tissue. A possible nonspecific cytotoxic effect of liposomes alone was excluded because yeast cell binding to lymph node tissue obtained from mice treated with liposome not containing the toxin (L-PBS) was essentially the same as yeast binding to lymph node tissues obtained from mice treated with PBS only. A time course experiment with lymph node tissues treated with the L-Cl2MDP preparation showed that macrophages were eliminated by 1 day following treatment and the phagocytic cells did not return for at least 10 days (data not shown). However, a 5-day treatment was chosen as previously described (9). The L-Cl2MDP effect on the macrophages, as monitored by immunostaining, showed that yeast binding is related to the presence of resident macrophages in the medullary and subcapsular sinus regions of the lymph node. The involvement of other phagocytic cells such as neutrophils in the binding seems unlikely because the neutrophil-specific MAb, SK-208, showed that L-CI2MDP did not affect their tissue population. Animals that received India ink in their hind footpads showed evidence of ink accumulation in the popliteal lymph nodes within 1 min after the injection (data not shown). By 15 min, the ink became densely concentrated in lymph node cells in the medullary and subcapsular sinus regions in a pattern that closely resembled both the MONTS-4 immunostaining of macrophages and the pattern of yeast cell adherence. The L-Cl2MDP and India ink experiments strongly support the hypothesis that C. albicans yeast cells selectively adhere to macrophages within the medullary and subcapsular sinuses of mouse popliteal lymph node tissue. The above results are not unique to mice of a single strain, sex, or T-cell competency. These experiments were of interest because others have reported that female mice are more resistant than male mice to disseminated candidiasis (24) and that congenitally thymic deficient mice are more resistant to acute-phase disseminated candidiasis than their normal littermates (6, 25). Although the pattern of yeast cell binding to lymph node tissue from nude mice was similar to the pattern for tissue from T-cell-sufficient animals, the


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number of yeast cells binding to tissue from nude mice was almost twofold greater than the number binding to tissue from normal mice. Further work is necessary to determine the relevance, if any, of this observation to mechanisms of host resistance against disseminated candidiasis. Fibronectin and laminin have been reported to play a role in the adherence of C. albicans to host vascular structures (20) via integrin-like molecules produced by C. albicans (4, 20, 26). Our results showed that the yeast cell binding was not affected by the fibronectin or laminin. These studies are in agreement with previous studies in which integrins and other known adhesion systems were found not to be involved in adherence of yeast cells to splenic marginal zone macrophages (18). Our studies indicate that C. albicans yeast cells bind to splenic and lymph node macrophages by a unique system. The binding distribution pattern in both tissues is similar to that described for a sialoadhesin expressed by macrophages in these tissue locations (29), and we are currently investigating this similarity. ACKNOWLEDGMENTS

We appreciate the expert technical assistance offered by Marcia H. Riesselman with some of the ex vivo assay techniques. We also thank Mark A. Jutila for generously supplying us with the monoclonal antibodies and Ginger Perry for teaching us the immunoperoxidase staining technique. This work was supported by grant A124912 from the National Institutes of Health. REFERENCES

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E. B. De Souza. 1990. Corticoprotein-releasing factor receptors in mouse spleen: identification of receptor-bearing cells as resident macrophages. Endocrinology 127:440-452.