MOLECULAR MECHANISMS OF CYTOKINE IMMUNOREGUIATION 
Kenneth J. Hardy, M.D., Ph.D., Assistant Investigator 
The cytokine network directly or indirectly mod- 
ulates, among other processes, inflammation, he- 
matopoiesis, bone and muscle growth, wound heal- 
ing, scar formation, tumorigenesis, and immune 
responsiveness. Interferon-7 (IFN-7) occupies a cen- 
tral role in that network as a potent modulator of 
both the affector and the effector limbs of the im- 
mune response. Although promising as a therapeu- 
tic agent for immunodeficiency states, infectious 
and neoplastic diseases, and autoimmunity, IFN-7 
has perhaps more often been evoked in the patho- 
genesis of such disorders. Aberrant expression of 
this cytokine has been implicated in acquired im- 
mune deficiency syndrome (AIDS), multiple sclero- 
sis, chronic granulomatous disease of childhood, 
and several inflammatory arthritides. This clinically 
relevant molecule is broadly interactive with other 
members of the cytokine system and, as demon- 
strated in Dr. Hardy's laboratory, can be influenced 
both positively and negatively by a variety of stim- 
uli, including other cytokines, as well as its own 
gene product. To facilitate an understanding of ge- 
netic regulation within the cytokine network as a 
whole, using the IFN-7 gene as a model, this labora- 
tory has identified human cellular systems, both 
cultured and primary, in which this gene can be in- 
teractively and differentially modulated. These cells 
are then examined with regard to cell surface trig- 
gers, proximal metabolic pathways, and the molec- 
ular structure and function of their IFN-7 genes. 
I. Definition of Cellular Systems Differentially Regu- 
lating IFN-7 Gene Expression. 
Several human T cell tumor lines (including 
Jurkat, Hut 78, and D4) as well as non-T cell lines 
(such as Raji and HeLa) have been characterized on 
the basis of their tissue-specific and/or differential 
expression of human IFN-7. More recently. Dr. 
Hardy's laboratory has begun isolating lymphocytes 
directly from normal human peripheral blood and 
fractionating them into total peripheral blood lym- 
phocytes (PBLs); erythrocyte rosette positive and 
negative; and CD4+, CD8+, CD45R+, CD29+, 
CD16+, CDI9+, and CD15+ cells. Such mononu- 
clear cell subsets have been analyzed for their regu- 
lated expression of IFN-7, interleukin-2 (IL-2), and 
other cytokines. Titratable modulation of IFN-7 
gene expression by various combinations of anti- 
gen-presenting cells (APCs), lectin-phorbol ester 
combinations, and/or cytokine-soluble factors, has 
provided insights into cytokine regulation impos- 
sible to obtain using tumor cell lines. Primary lym- 
phoid cells and their subsets, when used in transient 
transfection studies, have become an important and 
physiologically relevant system for the molecular ge- 
netic analyses of lymphokine gene regulation. 
II. Surface Triggers and Proximal Metabolic Path- 
ways Leading to T Cell Activation and Lymphokine 
Gene Expression. 
The metabolism of the major T cell membrane 
phospholipid, phosphatidylcholine (PC), has been 
investigated in Dr. Hardy's laboratory and demon- 
strated to be involved in T cell signal transduction 
pathways generally attributed to phorbol 12-myris- 
tate 13-acetate (PMA) (and/or APCs). Specific syn- 
thesis (turnover) of phosphatidylinositol (PI) and 
PC in Jurkat, total PBLs, and their subsets has been 
found to depend on stimulation with either phyto- 
hemagglutinin (PHA), PMA, or both. Under condi- 
tions of T cell activation and IFN-7 gene induction, 
turnover of both PC and PI are increased signifi- 
cantly. Neither neonatal cord blood lymphocytes 
nor "naive" (CD45R''^) lymphocytes expressed IFN-7 
upon T cell activation, but both demonstrated nor- 
mal PI and PC turnover patterns, suggesting that 
differential inactivity of the human IFN-7 gene oc- 
curs through mechanisms distal to these metabolic 
pathways. In a perhaps related observation, no ef- 
fects of APCs, IL-1, and leukotrienes (normal up- 
regulatory signals to IFN-7 expression) on PI or PC 
pathways were seen. 
III. Structural and Functional Analysis of IFN-7 
Gene Regulation. 
A. Positive effectors oflFN-ygene regulation. Several 
physiologic agents have been implicated as up- 
regulators of IFN-7 gene expression. Definitive evi- 
dence has now been obtained from Dr. Hardy's labo- 
ratory that IFN-7 can autosuperinduce its own gene 
expression in a dose-dependent fashion. Both higher 
steady-state levels of IFN-7 transcripts and enhanced 
secretion of its biologically active product were ob- 
served. Highly purified CD3^ cells, either alone or 
reconstituted to autologous monocytes, were poorly 
responsive in this regard, arguing against an auto- 
crine mechanism. This was in sharp contrast to the 
strong upregulatory response of these same cells to 
accessory stimulation with another cytokine, IL-2. 
Preliminary data point to an important role for cells 
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