Abstract
LPS induces the maturation and activation of dendritic cells in part by inducing toll-like receptor 4 (TLR4) and subsequent activation of specific signal transduction pathways (Medzhitov, Nat Rev Immunol., 2001). We and others have identified differential CB-DC versus APB-DC immune functional responses (Bradley/Cairo, Human Immunol., 2005). We have recently identified differential gene expression patterns in CB versus APB monocytes (Jiang/Cairo, J. of Immunol., 2004) and DC (Jiang/Cairo, Blood/ASH, 2004). Significant different gene expression patterns have been identified including cell surface molecules, cytokines/chemokines, signaling molecules, immune regulators and transcription factors. The differential expression of some of these genes may in part be responsible for the functional differences of CB versus APB DC. We therefore sought to examine quantitative differential protein expression patterns between CB and APB DC using proteomic tool (isotope-coded affinity tagging (ICATTM) followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS)). Briefly, Mo were purified from fresh CB or APB and cultured for 8 days with GM-CSF and IL-4 (immature DC (iDC)) and activated by LPS for mature DC (mDC). Equal amounts of protein (1 mg) obtained from total cell lysates were reduced and labeled with isotope by mixing 1 unit of light (control) and heavy (treated) cleavable ICAT reagent and subjected to avidin affinity chromatography. Offline fractions were collected, digested with trypsin and analyzed by automated reverse phase nanospray LC-MS/MS. The MS/MS acquired data was searched with SEQUEST against amino acid sequences in the UniProt protein database to identify specific proteins. Our preliminary data demonstrated approximately 25 proteins overexpressed and 51 proteins underexpressed (> or < 1.5 fold threshold, respective) in APB iDC versus CB iDC. Similarly, there was a wide range of overexpressed (45) and underexpressed (108) proteins in APB mDC versus CB mDC. Specifically, our results indicated several Zinc finger proteins (292, 221, 474, 578) were expressed higher in APB versus CB mDC (2 to 5 fold). Two Ras-related small G-proteins Rab-3C and Ran-BP2 demonstrated 1.4 and 1.3 fold higher expression respectively in APB versus CB mDC. We also identified that interleukin-4 precursor revealed a 7.7 fold higher expression levels in APB DC versus CB mDC. In contrast, significant lower protein expression levels were demonstrated in APB versus CB DC, particularly cell cycle regulators cyclin I (3 fold), cyclin G-associated kinase (2 fold), Rb-like protein 2 (4.35 fold), PKC theta (2 fold). The expression of zinc finger proteins has been implicated to be associated with monocyte differentiation (Yuan, J. of Cancer Res., 2001) and dendritic cell differentiation (Li, Neuron, 2004). Additionally, Rab and Ran, small G-proteins, have been shown to be important for cell morphology and motility (Schmidt/Hall, Ann. Rev. Cell &Devel. Biol.,1998). In summary, this study reveals for the first time the global proteomic differences between CB and APB DC and provides novel insights into their differential immune function between CB versus APB DC.
Differential protein expression patterns between planktonic and biofilm cells of Salmonella enterica serovar Enteritidis PT4 on stainless steel surface
