In vitro, Candida albicans releases the immune modulator adenosine and a second, high-molecular weight agent that blocks neutrophil killing.
We previously described a lyophilized supernatant from germinated Candida albicans that blocks human neutrophil (PMN) O2- production and degranulation stimulated by several PMN agonists but does not block stimulation by PMA. In studies to further characterize this Candida hyphal inhibitory product (CHIP), we noted several physicochemical parallels with the purine nucleoside adenosine (Ado). A Sephadex G-10 semipurified fraction of CHIP had an absorption peak near 260 nm, an apparent m.w. of less than 400, and was resistant to boiling and proteases. Maximally effective doses of CHIP (100 micrograms/ml) and Ado (100 microM) blocked 0.1 microM FMLP-stimulated O2- production by 76.8 +/- 4.1 and 81.7 +/- 4.8%, respectively. Ado deaminase, known to inactivate Ado, reversed inhibition by both Ado and CHIP. Results were comparable for the effect of CHIP and Ado on FMLP-stimulated beta-glucuronidase and lactoferrin release. Activation of the respiratory burst by opsonized C. albicans yeast was also inhibited by CHIP and Ado, but the extent of inhibition was less than for FMLP. At yeast:PMN ratios of 4:1, 10:1, and 40:1, CHIP inhibited O2- by -3.8%, 14.3%, and 12.8%, respectively; Ado blocked production by 32.9%, 24.2%, and 11.5%, respectively. The effect of CHIP and Ado on Candida killing by PMN was compared using two viability assays in each of four experiments. Ado (100 microM) had no effect on killing, although CHIP (100 micrograms/ml) inhibited killing in the MTT assay at 15 and 45 min by 81.6 +/- 6.3 and 24.7 +/- 6.2%, respectively; as assayed by CFU, CHIP inhibited killing by 34.1 +/- 6.2 and 10.3 +/- 2.5%, respectively. The ability of CHIP to inhibit killing was not affected by adding Ado deaminase, providing additional evidence that an Ado-like effect by CHIP is not essential for killing inhibition. Killing of opsonized Streptococcus pneumoniae was also inhibited in a concentration-dependent manner. Reverse-phase HPLC of the semipurified fraction revealed a peak, eluting identically to authentic Ado, which was eliminated by adding Ado deaminase. Ado content of the G-10 fraction was sufficient to account fully for the FMLP-inhibitory activity. The antikilling activity was resistant to boiling and proteases but was eliminated by mild periodation. Fractions eluting from a Sephadex CL6B column between 0.8 and 2.0 x 10(6) m.w. had increased sp. act. for killing inhibition. Sp. act. increased as carbohydrate content increased, but killing inhibition by various Candida cell wall constituents was absent to modest compared to inhibition induced by CHIP.(ABSTRACT TRUNCATED AT 400 WORDS)
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Evans Memorial Department of Clinical Research, University Hospital, Boston University Medical Center, MA 02118.
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D000241:Adenosine / Q000378:metabolism* / Q000494:pharmacology; D001770:Blood Bactericidal Activity; D002176:Candida albicans / Q000276:immunology* / Q000378:metabolism; D015550:Cell Degranulation / Q000187:drug effects; D003602:Cytotoxicity, Immunologic; D006801:Humans; D066298:In Vitro Techniques; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000276:immunology*; D010587:Phagocytosis; D013296:Streptococcus pneumoniae / Q000276:immunology; D013481:Superoxides / Q000378:metabolism
D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000241:Adenosine