Secretion of cytokines and chemokines by polarized human epithelial cells from the female reproductive tract.

BACKGROUND: Pro-inflammatory chemokines that attract and cytokines that activate immune cells contribute to normal physiological homeostasis in the female reproductive tract, and are needed to deal effectively with potential pathogenic microbes. Mucosal epithelial cells are capable of producing these factors that communicate with cells of the innate and adaptive immune systems. METHODS: Epithelial cells from Fallopian tube, endometrium and endocervix were isolated and grown to high transepithelial resistance in cell inserts from seven patients who had hysterectomies. Interleukin (IL)-8, IL-6, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha) and macrophage inflammatory peptide-1beta (MIP-1beta) were assessed by Luminex bead analysis or enzyme-linked immunosorbent assay (ELISA) in epithelial cell conditioned media from the apical and basolateral compartments. RESULTS: With the exception of MCP-1, the seven chemokines/cytokines constitutively produced by the polarized epithelial cells were preferentially secreted apically. A concentration pattern was found in all cases, with IL-8 and IL-6 produced in the greatest quantity. CONCLUSIONS: The concentrations of IL-8, IL-6, G-CSF and MCP-1 are similar to the levels found in reproductive tract fluids of patients with infection. The constitutive secretion and compartmentalization of large quantities of bioactive chemokines and cytokines provide additional evidence for the role of epithelial cells as gatekeepers of innate immune protection in the female reproductive tract.

Down-modulation of the costimulatory molecule, CD28, is a conserved activity of multiple SIV Nefs and is dependent on histidine 196 of Nef.

In this study Nef proteins derived from simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) were compared to assess their abilities to down-modulate the cell surface levels of the T-cell costimulatory molecule CD28. We demonstrate that in addition to Nef derived from the prototypic SIVmac239, Nef proteins encoded by the pathogenic SIVsmmPBj molecular clone and the SIVsmmB670 isolate also down-modulate cell surface CD28. In contrast, Nef proteins derived from HIV failed to down-modulate CD28. We have also identified H196 as a critical residue which influences the capacity of SIVmac Nef to down-modulate CD28. Nef derived from SIVmacJ5 failed to down-modulate cell surface CD28, whereas a Q196H substitution mutant of SIVmacJ5 Nef was able to down-modulate cell surface CD28. Conversely, substitution of H196 to Q196 in SIVmac239 Nef resulted in a mutant that had minimal effect on cell surface CD28 expression, despite retaining the capacity to down-modulate cell surface CD3epsilon. H196 lies immediately adjacent to a documented di-leucine endocytic motif and mutation of this motif also abrogated the ability of SIVmac239 Nef to down-modulate CD28. These findings demonstrate that down-modulation of the costimulatory molecule, CD28, and clonotypic TCR/CD3 complex are conserved attributes of SIV Nef.

The T-cell receptor zeta chain contains two homologous domains with which simian immunodeficiency virus Nef interacts and mediates down-modulation.

We have recently demonstrated that simian immunodeficiency virus (SIV) Nef binds to the zeta chain of the T-cell receptor (TCR), leading to its down-modulation from T-cell surfaces (I. Bell, C. Ashman, J. Maughan, E. Hooker, F. Cook, and T. A. Reinhart, J. Gen. Virol. 79:2717-2727, 1998). Using a panel of human as well as rhesus macaque TCR zeta cytoplasmic domain mutants, we have identified in this report two linear peptides in the cytoplasmic domain of TCR zeta which independently interact with SIV Nef. Each SIV Nef interaction domain was sufficient in the absence of the other for interaction with SIV Nef in a yeast two-hybrid assay. In parallel, we demonstrated that Nef down-modulation of CD8-TCR zeta fusion proteins containing full-length or truncated portions of the TCR zeta cytoplasmic domain occurs in transiently transfected 293T cells. Furthermore, using proteins expressed in Escherichia coli, a glutathione S-transferase-Nef fusion protein coprecipitated histidine-tagged portions of the TCR zeta cytoplasmic domain which contained SNID-1 or SNID-2. The peptides targeted by SIV Nef, YNELNL and YSEIGMKGERRR, are portions of the first and second of three immunoreceptor tyrosine-based activation motifs which are important in signal transduction, thymocyte development, and TCR biogenesis. These results demonstrate that SIV Nef binds to two distinct domains on TCR zeta in the absence of other T-cell-specific factors, and that interaction with either domain is sufficient to cause down-modulation of TCR zeta.