George W. Cox

Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages

Transforming growth factor‐β1 (TGF‐β) is secreted in a latent form consisting of mature TGF‐β noncovalently associated with its amino‐terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF‐β from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF‐β action. We have identified two events associated with latent TGF‐β (LTGF‐β) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF‐β concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon‐γ and lipopolysaccharide reportedly activate LTGF‐β via cell membrane–bound protease activity. We show through dual immunostaining of paraformaldehyde‐fixed macrophages that such physiological TGF‐β activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF‐β epitopes. The induction of TGF‐β immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF‐β activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF‐β and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF‐β activation provides an important tool for studies of its regulation. J. Cell. Physiol. 178:275–283, 1999.

Comparison of control of Listeria by nitric oxide redox chemistry from murine macrophages and NO donors: insights into Listeriocidal activity of oxidative and nitrosative stress

The physiological function of nitric oxide (NO) in the defense against pathogens is multifaceted. The exact chemistry by which NO combats intracellular pathogens such as Listeria monocytogenes is yet unresolved. We examined the effects of NO exposure, either delivered by NO donors or generated in situ within ANA-1 murine macrophages, on L. monocytogenes growth. Production of NO by the two NONOate compounds PAPA/NO (NH2(C3H6)(N[N(O)NO]C3H7) and DEA/NO (Na(C2H5)2N[N(O)NO]) resulted in L. monocytogenes cytostasis with minimal cytotoxicity. Reactive oxygen species generated from xanthine oxidase/hypoxanthine were neither bactericidal nor cytostatic and did not alter the action of NO. L. monocytogenes growth was also suppressed upon internalization into ANA-1 murine macrophages primed with interferon-gamma (INF-gamma) + tumor necrosis factor-alpha (TNF-alpha or INF-gamma + lipid polysaccharide (LPS). Growth suppression correlated with nitrite formation and nitrosation of 2,3-diaminonaphthalene elicited by stimulated murine macrophages. This nitrosative chemistry was not dependent upon nor mediated by interaction with reactive oxygen species (ROS), but resulted solely from NO and intermediates related to nitrosative stress. The role of nitrosation in controlling L. monocytogenes was further examined by monitoring the effects of exposure to NO on an important virulence factor, Listeriolysin O, which was inhibited under nitrosative conditions. These results suggest that nitrosative stress mediated by macrophages is an important component of the immunological arsenal in controlling L. monocytogenes infections.

Molecular Cloning and Characterization of a Novel Mouse Macrophage Gene That Encodes a Nuclear Protein Comprising Polyglutamine Repeats and Interspersing Histidines

Simple tandem repeats of the trinucleotide sequence CAG encode homopolymeric stretches of glutamine. Although polyglutamine has been identified in diverse proteins, it is present predominantly in transcription factors. We observed that oncogene-immortalized mouse macrophages express several genes that contain a CAG repeat motif. Therefore, we attempted to clone a novel gene that contains a CAG repeat and is associated with cytokine activation of macrophages. Screening of a mouse macrophage cDNA library with a probe comprising 12 consecutive CAG triplets identified at least one unique clone. The cDNA encodes a protein (named GRP-1 or lutamine epeat rotein-1) with 171 amino acids, a calculated molecular mass of 21.6 kDa, and a predicted pI of 10.67. Greater than two-thirds of GRP-1 are only two amino acids, namely glutamine (50%) and histidine (18%). There are four polyglutamine motifs interspersed with histidine-rich regions. There is also a putative nuclear localization signal flanked by sites for possible serine phosphorylation. GRP-1 mRNA was expressed constitutively in some macrophage cell lines and B and T cell lines. Interferon-γ or lipopolysaccharide augmented GRP-1 mRNA expression in the mouse macrophage cell line ANA-1. Western blot analyses using an antipeptide serum revealed that GRP-1 was localized in the nucleus of ANA-1 macrophages and transfected 3T3 fibroblasts. Overexpression of GRP-1 decreased Sp1-driven chloramphenicol acetyltransferase gene expression in transient cotransfection experiments. Because polyglutamine motifs can cause protein oligomerization and can function as transcriptional activation domains, we suggest that GRP-1 may be a transcription factor associated with interferon-γ- or lipopolysaccharide-induced activation of macrophages.

Characterization of nitric oxide–stimulated ADP‐ribosylation of various proteins from the mouse macrophage cell line ANA‐1 using sodium nitroprusside and the novel nitric oxide‐donating compound diethylamine dinitric oxide

We examined the ability of nitric oxide (NO) to stimulate the ADP‐ribosylation of proteins from the mouse macrophage cell line ANA‐1. To demonstrate a specific effect of NO, we used a novel compound named diethylamine dinitric oxide (DEA/NO; l,l‐diethyl‐2‐hydroxy‐2‐nitrosohydrazine, sodium salt; [Et2NN(O)NO]Na), which releases NO in aqueous solution at neutral pH. DEA/NO stimulated the ADP‐ribosylation of at least three cytosolic proteins (Mr = 28,000, 33,000, and 39,000) from ANA‐1 macrophages. The effect of DEA/NO on the ADP‐ribosylation of the predominant target p39 was dose dependent (EC50 = 80 μM). Moreover, the effect of DEA/NO was attributed specifically to released NO rather than diethylamine or nitrite. Sodium nitroprusside (SNP) also stimulated the ADP‐ribosylation of cytosolic proteins from ANA‐1 mouse macrophages. However, SNP exhibited different time‐ and dose‐dependent effects on the modification of p39. NO synthesized via the activity of interferon‐γ plus lipopolysaccharide‐induced NO synthase also enhanced the ADP‐ribosylation of p39, confirming that the effects of DEA/NO and SNP could be attributed to NO or reactive nitrogen oxide species. Neither pertussis toxin nor cholera toxin stimulated the ADP‐ribosylation of p39; however, cholera toxin stimulated the ADP‐ribosylation of proteins with approximate molecular weights of 28,000 and 33,000. These data suggest that the induced expression of NO synthase in tumoricidal macrophages may be associated with autocrine and paracrine effects of NO that include the ADP‐ribosylation of various proteins. Moreover, these results indicate that DEA/NO and related compounds may be useful as pharmacologic tools for investigating the effects of NO and reactive nitrogen oxide species on macrophages. J. Leukoc. Biol. 57: 152–159; 1995.

Synaptic and Extrasynaptic Action of Free Radicals on Cell-to-Cell Signaling

Nitric oxide (NO) is generated under physiological conditions and plays a role as a transducer in the central and peripheral nervous systems. In brain, the formation of N O from L-arginine is catalyzed by nitric oxide synthase (NOS), an enzyme present in certain neurons and glial cells. One of the mechanisms that leads to increased NO formation in NOS-containing neurons involves glutamate receptor stimulation that is associated with increased Ca2+-dependent inward currents. The rise of cytosolic Ca2+ brings about the Ca2+/calmodulin-dependent activation of NOS in postsynaptic neurons.z4 Recently, we reported that upon N-methyl-Daspartate (NMDA) receptor stimulation N O is synthesized in postsynaptic neurons. It then diffuses across the synaptic cleft and causes the release of neurotransmitters at presynaptic nerve terminals. The mechanism by which NO mediates neurotransmitter release is still unknown. In striatal slices inhibition of NOS by N-nitroarginine abates dopamine release mediated by NMDA receptor stimulation.s Furthermore, 3H-dopamine release is also attenuated by the N O scavenger, hemoglobin, supporting the concept that NO diffuses through extracellular space to reach its target site.s The finding that NO is a freely diffusable gas, makes it an interesting candidate for a potential retrograde neuronal messenger. Besides mediating the release of neurotransmitters,s N O also has been shown to regulate the release of hormonesh and releasing factors. Here, we summarize our recent work that demonstrates that N O is released by a Caz+-independent mechanism from axonal and (or) dentritic terminals, and propose that N O may play a novel role in the regulation of extracellular dopamine content. Such a role may have important implications in governing postsynaptic dopamine receptor occupancy involved in control of mood and behavior. In addition, evidence of an intricate morphological organization of dentrites from dopaminergic neurons around blood vessels in the substantia nigra8 suggest that the afferent stimulation by NO may also play an important role in vascular and microcapillary function in specific brain regions.