We have continued to study the cellular and molecular processes that control chemokine structure and function with an emphasis on chemokines that effect HIV-1 replication. Natural forms of chemokines secreted from normal lymphocytes and macrophages under different cell activation conditions have been purified from cells and culture supernatants using chemokine specific antibodies. The proteins are then analyzed using mass spectrometry and gel electrophoresis. Kinetic studies of chemokine secretion have revealed a complex pattern of secretion and protein processing. Chemokines secreted from T cells during cell activation initially are present intracellularly and then secreted as unprocessed proteins. Chemokines that carry a CD26 dipeptidase motif (RANTES and MIP- 1beta) then appear as truncated forms within 24-48 hours after activation consistent with the expression of CD26 on the cell membrane. Truncated forms of these chemokines show changes in receptor interactions compared with full-length forms, suggesting that protein processing through the CD26 dipeptidase system provides a mechanism to either inactivate or alter the activity of specific chemokines. Chemokines that are not sensitive to CD26 cleavage in vitro also undergo natural processing in cells demonstrating that several pathways of controlled proteolysis contribute to chemokine function in lymphocytes and macrophages. We believe that differential regulation of chemokine structure provides a molecular mechanism that controls the type of T cell (Th1 vs. Th2) recruited into an inflammatory site, which then determines the type of immune response generated to antigenic challenge. In addition to studies on chemokine processing, we have also investigated protein-protein interactions in native mixtures of chemokines. We have discovered that the chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta are predominately secreted from activated human monocytes and peripheral blood lymphocytes (PBLs) as heterodimers of these proteins. Immunoprecipitation and immunoblot analysis revealed that antibodies to either MIP-1alpha or MIP-1beta precipitated a protein complex containing both MIP-1alpha and MIP-1beta from culture supernatants of these cells. Mass spectrometry of the complexes precipitated from the culture supernatants of monocytes and PBLs revealed the presence of NH2- terminally truncated MIP-1alpha together with either intact MIP-1beta or NH2-terminally truncated MIP-1beta, respectively. The heterodimers form intracellularly in the Golgi complex before secretion. The secreted MIP- 1alpha/beta heterodimers were dissociated into their component monomers under acidic conditions. Mixing of recombinant chemokines in vitro demonstrated that heterodimerization of MIP-1alpha and MIP-1beta is specific and that it occurs at physiological conditions (pH7.4 and in the range of nM concentrations). The ability of the MIP-1alpha/beta heterodimer to induce down-regulation of the receptor CCR5, as monitored by flow cytometry, was greater than that of monomeric MIP-1alpha or MIP- 1beta. It is likely that formation of heterodimers of MIP-1alpha/beta has an impact on intracellular signaling events that may contribute to the enhanced activity of the mixture of MIP-1alpha and MIP-1beta on CCR5 modulation.
