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Phagocytosis of Candida albicans by eosinophilic leukocytes
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Phagocytosis of Candida albicans by eosinophilic leukocytes Takeru Ishikawa, Carl E. Arbesman, Hu~ulo, 3. I’.
M.D.,* M.D.
A. Condon
Dalton,
M.D.,*
and
The percentage of eosinophils phagocytosing Candida albicans spores approximates that of neutrophils in. in vitro etperiments. The phagocytic ncticity of the cosinuphils is shown to parallel anti-Candirla IgG serum antibody titers and to involve the complement system. By means of an immunofuorescent technique, with the use of rho&mine II conjugated to bovine serum albumin to block nonspccifi: sitcining, the presence of antibody on the phagocytosed spores is demonstrated.
The function of eosinophils in immunologic phenomena remains an enigma despite considerable investigation. Evidence has accumulated that these cells may phagocytosc antigen-antibody complexes,l-’ bacteria,“, D and mycoplasma.’ Lehrer has recently demonst.rated the ability of cosinophils to ingest and kill Candida albicans spores.8 This report confirms the ability of the human cosinophil to phagocytose C. albicms. In addition, the attachment of immunoglobulin G to the engulfed organisms is demonstrated by an immunofluorescent technique. MATERIALS
AND
METHODS
Normal human IgG was isolated from serum by DEAE-cellulose: c:hronmtography and thcw digested with papain. The digested product was fractionated Iby chromatography on Sephadex G.200, following which starch block electrophoresis was performed. The Fc fragment thus obtained was used to immunize rabbits, with the production of a monospecific antiserum. Partly purified IgA, IgN, and IgD, isolated from myeloma sera, were used to immunize rabbits. After the animals were bled, monospecific antisera were obtained by albsorptiou with humnn cord blood which had bcrn tropolymrrized with glutaruld(*hydf~. .\ionospwific goat antiserum to human IgE was obtained by a method prrviously describeda These antisera were conjugated with fluorescein isothiocyanatc (FITC), and the free FITC From the State University of New York at Buffalo, and the Allergy Rescnrch Laboratory of the Buffalo General Hospital. Supported in part by United States Public Health Service Research Grant 5.ROl-AI.01303 and in part by United States Public Health Service Training Grant 5TOl-Al-00051. Reprint requests to: Dr. Arbesman, Allergy Research I.al&tory, 100 High St., Buffalo, s. Y. 11203. Received for publication Sept. 13, 1971. ‘Rwipients of the 1)r. Henry C. Buswell and Bertha Ii. Buswcll Rcwarch E’cllownhip. IrJ.
49. so.
5, pp. 31 1 -.?lB
%
%
r: - - -
1.
% 7.
%
-. f._.- ^ &-, ..T. _ ‘, _. ,-
%
VOLUME NUMBER
49 5
Phagocytosis
L
of
Candida
albicons
313
---I----I---, undil
1:3
1:9
1:27
Wi
1243
t729
la87
l656l
Serial Dilution of Serum 1. The parallel spores by eosinophils and &, 125. 0 = eosinophils. FIG.
relationship of serum anti-Candid0 and neutrophils is demonstrated. serum 1; 0 = serum 2; 0 =
IgG
Titer serum
titers
and
of
serum
3;
N
=
phagocytosis
S, is 5; neutrophils;
s,
of
729; E
=
are prcscntcd graphically in Fig. 1. By comparing these results with the antibody titers recorded in Table I, it can bc seen bhat there is a correlation between the degree of phagocytosis and the anti-Candida IgG titers. Such an association is not apparent with Igil ant1 1gId. When serum was not incubated with organisms or when the serum lacking antibod~~ (S,,) was usetl, the percentage of eosinophils containing organisms dicl not exceed 13.5, and the percentage of neutrophils tlicl not exceed 1 2.5. This low Agree of phagocytosis is probabl: accounted for by the inability to coruplctely wash off antibody from the donor leukocytes, for the fluorosc!ent staiIting rrvealetl lg4: antibody attached to phagocytosecl organisms. Over all, when invubatecl with the 6 sera containing I#; antibocl>-, the aycragc number of cosinophils mltainiug organisms was 55.8 per cent., closely approximating tlir number of nrntrophils (51.5 per cent). ITcating the srra at 56O C. for 30 miuuirs hrfo~ incubation decreased the degree of phagoqtosis three- to sixfoltl. In cvctry preparation, alniost all Cantlida organism scrn within cells stainetl positively with the PlTC-anti-IgC conjugat.o (Fig. 2). The great majority, but not all, of phagocytosrtl spores stained with the anti-p,(l (*onjllg;~t(~. DISCUSSION Pliagocytosis of (,‘. rrbhictf)ts sl~ores by human eosinophils is clearly tlcnionstrilt(d ill this study, and the prrcentagv of Wiillophils engaging in ljhagotrytosis c~loscly approximates that ot’ the neutrophils. Two ill(lictitims ol’ t.hcb importiLtl(ae of :lntigcIn-aIltihotl?mnplcxvn in this ~)I~PIIOI~I(‘IIOII ilI*e 11ot(v1. Thtb first is that. ;llnlost illI orgiltlisms svvn withill cv~sitlophils stainvtl with t*iilJl)it anti hutt11111I@ ~o~i,jtigalcd with E’ITC!. Scco~itlly, the nlJility of strum to cnhancv
314
FIG. 2. eosinophil
Ishikowo,
The
Dalton,
attachment is shown by
and
ALiERGY
Arbesman
of IgG antibody immunofluorescent
to o C. technique.
albicans
spore
contolned
CLlN
IMMUNO: MAY !972
within
on
VOLUME NUMBER
49 5
Phagocytosis
of
Candida
albicans
315
REFERENCES 1 Sabesin, S. E.: A function of the eosinophil: Phagocytosis of nntigen.antibody COnl~ plexus, Proc. Sot. Exp. Biol. Med. 112: 667. 1963. .4nn. N. T. Acnd. Bci. 116: 964, 196-l. 2 Litt, M.: Eosinophils and antigenantibody reactions, granule IFsis in vitro induced 3 Archer, Cr. T.. Nelson, hf., and Johnston, J.: Eoeinophil 11~ solul~lc xntig(,n-:lntil)oclv complexes, Immunology 17: 177, 1969. C. E. : Tn vitro am1 iu rive ndllcsion 4 lsl~ikaw:~, T., Evans, R., \Vicher, K., and Arbesman, of :intipen.antibodp compleses to cosinophils, Fed. Proc. 36: 2694, 1971. (AM.) 5 Strumia, hT.M., and Bocrnor, F.: Phagocytotic activity of circulating cells in tllv various types of leukemia, Am. J. Path. 13: 335. 1937. 6 ( ‘ol~rw, S. G.? and Sxpp, T. 11.: Phagocytosis of bacteria by eosinophils in infecliouswhited asthma, J. .-\I.I.ERGY 44: 113, 1!#69. 7 %uck~,r.Fr:lllklirl, I)., Davidson, hr., and Thomas, L. : The interaction of myq~lasnu~s witli nl:tnIux~li:lu cells. T. Hela cells, neutrophils ant1 eosinophils. J. E:xp. Med. 124: 521, 1966. 8 ln~hror, H. T.: hfeasuremcnt of cnntlidacidal activity of specific Icukocytc typos in mixed wll populations. II. Normal and chronic granulomatous disease eorinophils, Tnfwt. Jnmunol. 3: 800, 1971. 9 J to, K., \Vicher, K., and Arlwsmxn, C. E.: Studies of TgE fragments prepare~l hy lmlmiu tligextion, lnt. Arch. Sllergp Appl. Immunol. 41: 4ii. 19il. 10 Ihwtncr, E. H., Yepulrcda, M. H., and Barnctt, E. I-.: Qmntitativc studies of inmlunofluorcsccnt staining, Bull. 1V.H.O. 13: 38i, 1968. 11 lshikawa, T., \\‘icher, I<., and Arbesmnn, C. R.: Eosinophilic Icukocytc~s and antigen antibody complexes, J. AT.I.ERGY 47: 101, 1971. (AM.) 12 l;sterlJ-, S. I~.: Serum antibody titers to Cnatlrdct rtlbiwns utilizing an illlmutlofluor(,sc.eIlt twhnique, Am. J. Clin. Pnthol. 50: 291, 1968. 13 I’anush, R. S., France, A. E., and Pchur, P. H.: Hht~un~atoitl arthritis nssoc~iaietl with wsinophilia, Ann. intern. Med. 75: 199, 197 I.
Phagocytosis of Candida albicans by eosinophilic leukocytes Takeru Ishikawa, Carl E. Arbesman, Hu~ulo, 3. I’.
M.D.,* M.D.
A. Condon
Dalton,
M.D.,*
and
The percentage of eosinophils phagocytosing Candida albicans spores approximates that of neutrophils in. in vitro etperiments. The phagocytic ncticity of the cosinuphils is shown to parallel anti-Candirla IgG serum antibody titers and to involve the complement system. By means of an immunofuorescent technique, with the use of rho&mine II conjugated to bovine serum albumin to block nonspccifi: sitcining, the presence of antibody on the phagocytosed spores is demonstrated.
The function of eosinophils in immunologic phenomena remains an enigma despite considerable investigation. Evidence has accumulated that these cells may phagocytosc antigen-antibody complexes,l-’ bacteria,“, D and mycoplasma.’ Lehrer has recently demonst.rated the ability of cosinophils to ingest and kill Candida albicans spores.8 This report confirms the ability of the human cosinophil to phagocytose C. albicms. In addition, the attachment of immunoglobulin G to the engulfed organisms is demonstrated by an immunofluorescent technique. MATERIALS
AND
METHODS
Normal human IgG was isolated from serum by DEAE-cellulose: c:hronmtography and thcw digested with papain. The digested product was fractionated Iby chromatography on Sephadex G.200, following which starch block electrophoresis was performed. The Fc fragment thus obtained was used to immunize rabbits, with the production of a monospecific antiserum. Partly purified IgA, IgN, and IgD, isolated from myeloma sera, were used to immunize rabbits. After the animals were bled, monospecific antisera were obtained by albsorptiou with humnn cord blood which had bcrn tropolymrrized with glutaruld(*hydf~. .\ionospwific goat antiserum to human IgE was obtained by a method prrviously describeda These antisera were conjugated with fluorescein isothiocyanatc (FITC), and the free FITC From the State University of New York at Buffalo, and the Allergy Rescnrch Laboratory of the Buffalo General Hospital. Supported in part by United States Public Health Service Research Grant 5.ROl-AI.01303 and in part by United States Public Health Service Training Grant 5TOl-Al-00051. Reprint requests to: Dr. Arbesman, Allergy Research I.al&tory, 100 High St., Buffalo, s. Y. 11203. Received for publication Sept. 13, 1971. ‘Rwipients of the 1)r. Henry C. Buswell and Bertha Ii. Buswcll Rcwarch E’cllownhip. IrJ.
49. so.
5, pp. 31 1 -.?lB
%
%
r: - - -
1.
% 7.
%
-. f._.- ^ &-, ..T. _ ‘, _. ,-
%
VOLUME NUMBER
49 5
Phagocytosis
L
of
Candida
albicons
313
---I----I---, undil
1:3
1:9
1:27
Wi
1243
t729
la87
l656l
Serial Dilution of Serum 1. The parallel spores by eosinophils and &, 125. 0 = eosinophils. FIG.
relationship of serum anti-Candid0 and neutrophils is demonstrated. serum 1; 0 = serum 2; 0 =
IgG
Titer serum
titers
and
of
serum
3;
N
=
phagocytosis
S, is 5; neutrophils;
s,
of
729; E
=
are prcscntcd graphically in Fig. 1. By comparing these results with the antibody titers recorded in Table I, it can bc seen bhat there is a correlation between the degree of phagocytosis and the anti-Candida IgG titers. Such an association is not apparent with Igil ant1 1gId. When serum was not incubated with organisms or when the serum lacking antibod~~ (S,,) was usetl, the percentage of eosinophils containing organisms dicl not exceed 13.5, and the percentage of neutrophils tlicl not exceed 1 2.5. This low Agree of phagocytosis is probabl: accounted for by the inability to coruplctely wash off antibody from the donor leukocytes, for the fluorosc!ent staiIting rrvealetl lg4: antibody attached to phagocytosecl organisms. Over all, when invubatecl with the 6 sera containing I#; antibocl>-, the aycragc number of cosinophils mltainiug organisms was 55.8 per cent., closely approximating tlir number of nrntrophils (51.5 per cent). ITcating the srra at 56O C. for 30 miuuirs hrfo~ incubation decreased the degree of phagoqtosis three- to sixfoltl. In cvctry preparation, alniost all Cantlida organism scrn within cells stainetl positively with the PlTC-anti-IgC conjugat.o (Fig. 2). The great majority, but not all, of phagocytosrtl spores stained with the anti-p,(l (*onjllg;~t(~. DISCUSSION Pliagocytosis of (,‘. rrbhictf)ts sl~ores by human eosinophils is clearly tlcnionstrilt(d ill this study, and the prrcentagv of Wiillophils engaging in ljhagotrytosis c~loscly approximates that ot’ the neutrophils. Two ill(lictitims ol’ t.hcb importiLtl(ae of :lntigcIn-aIltihotl?mnplcxvn in this ~)I~PIIOI~I(‘IIOII ilI*e 11ot(v1. Thtb first is that. ;llnlost illI orgiltlisms svvn withill cv~sitlophils stainvtl with t*iilJl)it anti hutt11111I@ ~o~i,jtigalcd with E’ITC!. Scco~itlly, the nlJility of strum to cnhancv
314
FIG. 2. eosinophil
Ishikowo,
The
Dalton,
attachment is shown by
and
ALiERGY
Arbesman
of IgG antibody immunofluorescent
to o C. technique.
albicans
spore
contolned
CLlN
IMMUNO: MAY !972
within
on
VOLUME NUMBER
49 5
Phagocytosis
of
Candida
albicans
315
REFERENCES 1 Sabesin, S. E.: A function of the eosinophil: Phagocytosis of nntigen.antibody COnl~ plexus, Proc. Sot. Exp. Biol. Med. 112: 667. 1963. .4nn. N. T. Acnd. Bci. 116: 964, 196-l. 2 Litt, M.: Eosinophils and antigenantibody reactions, granule IFsis in vitro induced 3 Archer, Cr. T.. Nelson, hf., and Johnston, J.: Eoeinophil 11~ solul~lc xntig(,n-:lntil)oclv complexes, Immunology 17: 177, 1969. C. E. : Tn vitro am1 iu rive ndllcsion 4 lsl~ikaw:~, T., Evans, R., \Vicher, K., and Arbesman, of :intipen.antibodp compleses to cosinophils, Fed. Proc. 36: 2694, 1971. (AM.) 5 Strumia, hT.M., and Bocrnor, F.: Phagocytotic activity of circulating cells in tllv various types of leukemia, Am. J. Path. 13: 335. 1937. 6 ( ‘ol~rw, S. G.? and Sxpp, T. 11.: Phagocytosis of bacteria by eosinophils in infecliouswhited asthma, J. .-\I.I.ERGY 44: 113, 1!#69. 7 %uck~,r.Fr:lllklirl, I)., Davidson, hr., and Thomas, L. : The interaction of myq~lasnu~s witli nl:tnIux~li:lu cells. T. Hela cells, neutrophils ant1 eosinophils. J. E:xp. Med. 124: 521, 1966. 8 ln~hror, H. T.: hfeasuremcnt of cnntlidacidal activity of specific Icukocytc typos in mixed wll populations. II. Normal and chronic granulomatous disease eorinophils, Tnfwt. Jnmunol. 3: 800, 1971. 9 J to, K., \Vicher, K., and Arlwsmxn, C. E.: Studies of TgE fragments prepare~l hy lmlmiu tligextion, lnt. Arch. Sllergp Appl. Immunol. 41: 4ii. 19il. 10 Ihwtncr, E. H., Yepulrcda, M. H., and Barnctt, E. I-.: Qmntitativc studies of inmlunofluorcsccnt staining, Bull. 1V.H.O. 13: 38i, 1968. 11 lshikawa, T., \\‘icher, I<., and Arbesmnn, C. R.: Eosinophilic Icukocytc~s and antigen antibody complexes, J. AT.I.ERGY 47: 101, 1971. (AM.) 12 l;sterlJ-, S. I~.: Serum antibody titers to Cnatlrdct rtlbiwns utilizing an illlmutlofluor(,sc.eIlt twhnique, Am. J. Clin. Pnthol. 50: 291, 1968. 13 I’anush, R. S., France, A. E., and Pchur, P. H.: Hht~un~atoitl arthritis nssoc~iaietl with wsinophilia, Ann. intern. Med. 75: 199, 197 I.