Measurement of cytokines and chemokines in parotid saliva from HIV+ patients

Abstract

Objective. Salivary cytokines and chemokines may reflect the local immunity to microorganisms as well as any ongoing salivary gland dysfunction. In the past, cytokine profiles in human whole saliva have been proposed to assess oral mucosa immunity during HIV infection. Studies have shown that alterations in the expression of cytokines in glandular saliva are associated with candidal infection in HIV patients. Currently, an extensive cytokine and chemokine profile in saliva has not been established. Recent development of a multiplexed fluorescent microsphere-based immunoassay (xMAP technology) allows quantifying multiple proteins simultaneously using a very small amount of sample. The purpose of this study is to (1) establish a multiplexed assay using xMAP technology to measure cytokines and chemokines in saliva samples and (2) compare salivary cytokine and chemokine profiles between saliva samples from healthy subjects and HIV(+) patients. Study design. The Luminex 100 system is an xMAP technology and consists of a 10 × 10 matrix with a 100-analyte capacity. A set of cytokine and chemokine antibody–coupled fluorescent beads was used for the quantification of citrate-stimulated parotid saliva from HIV+ (n = 4; CD4+ counts < 200 cells/μL; salivary Candida count 500-9,000 cfu/mL) and healthy HIV− (n = 4; Candida 0 cfu/mL) subjects. The cytokines—interleukins (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL10, IL-12, IL-13, and IL-15), interferon (IFN-γ), TNF-α, and GM-CSF—and the chemokines Eotaxin, RANTES, MCP-1, and MIP1α were assayed using 10 μL of saliva. Results. All 16 cytokines and 4 chemokines studied could be measured in detectable levels (>1 pg/mL). Overall, parotid saliva showed high concentrations of IFN-γ and Eotaxin (>200 pg/mL) and low concentrations of IL-1β, IL-13, and GM-CSF (<10 pg/mL). The concentrations of Eotaxin, GM-CSF, IL-1α, IL-1β, IL-2, IL-12p40, and IL-12p70 were elevated in stimulated parotid saliva from HIV+ patients compared to healthy HIV− subjects. The profiles for pg/mg total protein are similar to concentrations. Furthermore, 2 patients with abnormally low saliva secretions (<0.2 mL/min) had higher IL-2 and GM-CSF levels compared to patients with normal secretions (>0.2 mL/min). Conclusions. This is the first report that analytes can be measured in saliva using a multiplexed fluorescent microsphere-based platform, indicating that xMAP technology can assess salivary cytokine profiles. Although the number of subjects studied was small, the trends observed in this study suggest different salivary cytokine profiles for patients with HIV infection with a significant oral candidal burden and HIV− controls. The elevated levels of inflammatory cytokines and chemokines in saliva from HIV+ patients may reflect an ongoing immune response to a pathogen or microorganism. The increased levels of IL-2 in patients with low saliva production suggest the presence of T lymphocytes in the parotid gland.