T cells tell macrophages when to start making the toxic soup of lysosomal enzymes, reactive oxygen species, and nitric oxide that destroys intracellular pathogens. In 1983, Carl Nathan proved that this start signal comes in the form of the secreted cytokine IFNγ.
1). But exactly how T cells turned the passive macrophages into aggressive killers remained a mystery for more than a decade.
The elusive eluate
Nathan, as an oncology fellow, knew well that white blood cells fought infections. “This was an experiment repeated in front of us all the time,” he says: chemotherapy lowered his patients' white blood cells and increased their risk of infections.
One link in that infection-fighting chain was T cell activation of macrophages. Mackaness had shown that macrophage activation did not depend on direct contact with T cells (1), suggesting the possibility of a secreted factor. When Nathan tested the supernatant from activated T cells, he saw that it did indeed induce macrophage activation (2).
The race was on to purify and identify the mystery factor. Nathan got a rough idea of the molecular weight (3), but that was “the best anyone could do,” he says. Protein separation methods were primitive, and cloned proteins and monoclonal antibodies would only become available a decade later. Henry Murray, one of Nathan's collaborators, sums up the feeling of frustration: “We were all nibbling at the edges of the same problem.”
Nathan therefore changed tack to take a closer look at the activated macrophages. Short-lived neutrophils were known to produce hydrogen peroxide, and Nathan found the same was true of longer-lived activated macrophages (4). Unlike previous signs of macrophage activation—increased spreading, phagocytosis, and glucose metabolism— this so-called “respiratory burst” correlated with the cells' ability to kill intracellular parasites (4).
Back on track
Meanwhile, candidate macrophage activators were in the news. IFNα had been on the cover of Time magazine, and recombinant murine IFNγ was found to induce macrophages to kill tumor cells (5). Nathan, now a faculty member in Zanvil Cohn's “macrophage factory” at Rockefeller University (New York, NY), thought IFNγ might also activate macrophages to kill intracellular parasites.
Consistent with this idea, IFNγ was made by antigen-stimulated T cells and was associated with defense from infection. Now the respiratory burst gave Nathan an assay, Berish Rubin (down the street at the New York Blood Center) supplied an IFNγ monoclonal antibody, and a phone call to Genentech yielded recombinant IFNγ. In a seminal paper published in The Journal of Experimental Medicine in 1983, Nathan was thus able to show that depleting IFNγ from unpurified T cell supernatants decreased the respiratory burst activity and the killing of intracellular protozoa in human macrophages. Adding back recombinant IFNγ into this mix restored macrophage activation (6).
The clues were there, says Nathan, but to be honest “I would have tried any cytokine that was purified to homogeneity. I had an assay, a hunch, a history of purifying proteins that did this, and the serendipity of meeting with people nearby who had the antibody.”
Nathan next showed that IFNγ worked in people. Injecting recombinant IFNγ directly into cutaneous lesions of lepromatous leprosy patients induced macrophage infiltration, hydrogen peroxide production, and killing of the causative pathogen, Mycobacterium leprae (7). In the 1990s, the macrophages of children with IFNγ receptor deficiencies were shown to be defective in killing mycobacteria (8). Tracing the pathway from T cells to macrophages to bacteria started, for Nathan, in 1967, and he says “we still haven't finished making the molecular links.”