It was actually accidental. When I became an immunologist, I found all these mediators that are now called cytokines and are analogous to hormones of the endocrine system. I was working on cytokines like interleukin-1 for a number of years, as a pioneer in the field. Then many other labs started to work on it, as well. Now I had to read all these other people’s papers just to keep up to date. I also realized that I couldn’t compete with hundreds of other labs. So, luckily, I found these contaminants in the interleukin-1 supernatant preparations that, when purified, turned out to be interleukin-8 and MCP1, respectively. These are members of the chemokine group of families.

“The notion that these antimicrobial chemokines have another function, mobilizing immune cells, is a big deal.”

There are now over 40 different chemokines. You can think of these as the traffic cops in the body: they direct inflammatory cells and lymphocytes involved in the development of lymphoid systems. I was working with interleukin-8, and I knew it to be an attractant to cells called neutrophils—cells that are involved in acute inflammation. This is what pus is in an abscess. I injected it under the skin of mice to make sure that what I had was active, and then I looked and, indeed, early on, at about four hours I got a lot of neutrophils at the site of the injection. I injected it daily, and I kept looking, and by 72 hours the site of injection was loaded with mononuclear cells, consisting of monocytes and lymphocytes. So I asked, "How is this possible?"

Here’s interleukin-8—it’s supposed to attract neutrophils, and it does so in the beginning. Then the injection site gets converted from a neutrophil lesion to a mononuclear lesion. So I assumed it’s probably the case that the neutrophils are making something that then attracts these mononuclear cells—a sequential effect. I had a Russian biochemist who joined my lab, Oleg Chertov, and I asked him to do me a favor: to isolate from neutrophils in culture a mediator that’s able to activate and attract mononuclear cells? He had this tissue culture set up and he did a progressive sequential purification and separation of molecules, and he kept testing it for activity, and finally it came through and he said, "What I have, Dr. Oppenheim, has a 3,500 molecular weight." I said, "That’s crazy. Nothing is 3,500 molecular weight." All the chemokines are around 8,000 to 14,000. But he said there were these things called defensins, and these were 3,500 daltons.

I guess this happened during 1995. Anyway, I said, "What are defensins?" We looked them up, and they were these anti-microbial peptides. They’re present in neutrophils and skin cells and gut cells. Wherever we face the outside world, there are defensins of all kinds, including alpha and beta defensins. They are our homemade antibiotics; they kill bacteria at relatively high concentrations. They dissolve and punch holes in the membranes of the bacteria. They work like neomycin and other antibiotics. They were initially discovered and studied by Thomas Ganz and Bob Lehrer, about a decade prior to when we stumbled on them.

Yes. We didn’t know anything about defensins. The defensins we found were chemotactic for mononuclear cells, and we figured there must be some receptor on the cells with which they interact to produce this chemotactic mobilizing of cells, to attract the cells and make them move in a certain direction. So we published a paper on this and, of course, the reviewers gave us a terrible time. "Well, in your lab almost everything is chemotactic," they said. "What’s another chemotactic substance? Big deal. It’s unimportant." So I got very annoyed and thought if this stuff is important, then it ought to do something directly to the immune system. We worked on these defensins for about five years before we published that ‘99 paper that identified the chemokine receptor CCR6 as a receptor for beta-defensins as well and got us so many citations (Yang D, et al., "Beta defensins: linking innate and adaptive immunity through dendritic and T cell CCR6," Science 286[5439]: 525-8, 15 October 1999).

We simply mixed the defensin with an antigen and injected them together into mice. We found if we did that, the immune response to the antigen was much greater. And we had trouble publishing this, too. This was an old-fashioned experiment, just phenomenology. We didn’t know any of the mechanisms. We just said this was an observation showing that defensins have this in vivo ability to augment the immune response. It took us seven journals to get it published. Finally the International Journal of Immunology, a Japanese journal, felt sorry for us. This kind of thing is ridiculous. You can end up tearing your hair out.

So we were working on other chemokines at the time—interleukin-8, in particular—and the receptors had been identified for these chemokines. Some of them are actually entry points for HIV infection. So these receptors play all kinds of important roles. I got the idea that maybe defensins were using the chemokine receptor to make immune cells migrate. I forgot why I thought CCR6, which is one of these receptors, was a good candidate. I guess I didn’t know too many receptors at the time. Anyway, we had a morning lab session, and I suggested to Dr. Yang, who is first author on that paper, that maybe he should look at CCR6 and see if CCR6-positive cells will react and maybe CCR6-negative cells won’t. We did that experiment and it worked. We could then show that a bona fide chemokine for CCR6 could compete with defensins for the receptor and vice versa. And that established the specificity of the reaction.

That’s it. And it was accepted in Science. Of course, not without a struggle, but at least we didn’t have to go to seven other journals.

Well, it said that these antimicrobial defensins had an immune role and that they used an immune receptor expressed on host immune cells, which affected the immune response. That’s what was crucial. Not long afterward, we looked at another antimicrobial, cathelicidin, and we found that it uses another related receptor.

The notion that these antimicrobial chemokines have another function, mobilizing immune cells, is a big deal. And when they do this, they act at concentrations that are one hundredth the level at which they act as antimicrobials. They’re quite potent. Nano-molar concentrations of these defensins and cathelicidin are able to interact with receptors and mobilize inflammatory cells. You need micromolar or microgram quantities of these to lyse bacteria. But a teeny weeny amount of these will slip into the circulation or the tissues and can galvanize an immune response. That’s why this discovery is important.

Well, the story is still evolving, as most stories do. And I think there are probably a number of additional molecules, some of which we’re working on, that also participate in this alarmin activity. Alarmins typically act on antigen-presenting cells and T-cells—T-lymphocytes. The antigen-presenting cells are attracted by alarmins to sites, where they eat antigens. They chop them up and present them to lymphocytes, which then react. This is the way alarmins facilitate the immune response, by acting on antigen-presenting cells predominantly. The concept is that we have a number of molecules that are readily available in the body for galvanizing a rapid response to dangerous stimuli, for alerting the immune and host defense systems, accelerating our response to microbial invasion and injury, and these alarmins help initiate these immune reactions.

Well, the last review group at NIH that looked at this said that the defensin activity I’m involved in hasn’t been as big a hit, so maybe my lab should be closed.

I think it’s still expanding. It takes time to see what’s going on. Of course, all scientists think that what they’re doing is the greatest thing in the world, and who cares what the other guys are doing—so if a scientist tells you that what he’s doing is the greatest thing in world, so what? But amplification of a new field takes a while.

What’s most interesting as a possibility here is that we have these alarmins in our body, and when we mix them with antigens, they promote and augment the immune response to antigens. So in an immediate practical sense, they are potential vaccine adjuvants. Up to now the only thing used in humans as an adjuvant is aluminum hydroxide. When you get immunized for tetanus, they usually do it with aluminum hydroxide, which is a relatively modest adjuvant. I think these peptides and proteins which are our own alarmins may turn out to be more active, more potent adjuvants. My overall aim is hope this will end up being practical.

This review committee indicated that they didn’t think the research was all that impressive. This was two and a half years ago. I obviously don’t believe that is a correct evaluation. But it will take time for it to be considered important. The ball is rolling. That’s my job at the NIH, to get things going, until other researchers catch on and pitch in to accelerate progress in the field of alarmin research.

Joost J. Oppenheim, MD
Chief of the Laboratory of Molecular Immunoregulation
NCI-Frederick
Frederick, MD, USA