The Phagocyte Oxidase: The Early Years

Abstract

This autobiographical dialogue describes the scientific interchanges during the 1970s of the Boston-based investigators studying the respiratory burst associated with phagocytosis. The early studies to characterize the responsible enzyme are reviewed and placed in the context of the initial studies conducted by Bernard Babior and John Curnutte that led to identification of superoxide as the product of oxygen reduction generated by phagocyte activation. The posit of NADPH as the electron donor contested other co-factors, a hypothesis that proved correct. By the end of the decade (following elucidation of artefacts, demonstrating flavin dependence, and discovering the absence of a b-cytochrome in X-linked chronic granulomatous disease patients), the basic enzymatic architecture that produced reactive oxygen species mediating the inflammatory response had been elucidated.

Appendix: The Phagocyte in Immunology History

The story of the NADPH oxidase originates at the end of the nineteenth century. A new formulation of the relationship between host and contagious disease was formally stated in 1883 by Ilya Metchnikoff’s convergence of three disparate and thus far unrelated streams: (1) bacteria as etiologic agents of infection, (2) the nature and role of inflammation, and (3) the place of evolutionary principles as applied to physiology.⁷ The germ theory of disease was established by Louis Pasteur and Robert Koch by the mid-1870s, but there was no theory akin to our modem notion of immunological defense. Pasteur as late as 1880, while develo** vaccines, believed that immunity was conferred by exhaustion of essential nutrients, analogous to the test tube model systems of bacterial growth. Koch was not even interested in the host response, confining himself to the establishment of bacterial etiology. Inflammation was generally viewed as a deleterious process, whose various components were regarded as reactive, not defensive. The white cells, already identified as amoeboid phagocytes, with purposeful movement and containing bacteria, were dismissed as transport vehicles for the pathogens, with no protective function hypothesized. In short, how bacteria might cause disease, and more fundamentally, the relation of host and pathogen from a physiological (organism) or evolutionary (species) perspective was left mute.

At this early stage of immunology, Metchnikoff, an embryologist, proposed that mesodermic phagocytes, which in primitive organisms served a nutritive function, in higher animals with a digestive cavity, assumed new functions, devoid of their original digestive purpose. He extended the metaphor of “eat or be eaten” to a dedicated function of these cells, which now wandering beneath epithelial surfaces and various interstices, recognized foreign elements and devoured them. Originally, he viewed the process as a general physiological mechanism, which he called “physiological inflammation,” for the phagocytes in protecting the host, recognized the ‘other’ in every form—from senescent, malignant, damaged, or otherwise diseased cells, to foreign invaders. The latter became his focus only as he was drawn into vociferous debate with “pathologists” (microbiologists) and early immunochemists, who were by then fully engaged in establishing the physics and chemistry of life processes [91, 93, 94]. The issue focused on what they saw as Metchnikoff’s portrayal of the phagocyte as an independent agent, possessing primitive independent volition and thereby he was condemned as invoking vitalism to explain immunity.⁸ And here the oxidase story begins.

In their search for chemical mechanisms, the immunochemists of Metchnikoff’s day were preoccupied with exorcising mysterious, unaccounted forces that would compromise their aspirations for establishing a physics of life. Metchnikoff became a focal point of dispute because he described the phagocyte as exhibiting autonomous behavior. The cells seemingly ‘knew’ where to go (chemotaxis) and once at the site of damage or invasion they undertook the ‘responsibility’ of protecting the host organism by eating everything in their target range. The chemists would have none of it and in criticizing the absence of defined mechanisms they sought a pre-arranged chemical basis for host defense. They soon identified antibody and complement as chemical anti-bacterial substances and by 1908, when Metchnikoff shared the Nobel Prize with Paul Ehrlich (the leading immune-chemical theoretician), the course of twentieth century immunology was set on the chemical mechanisms of host defense.

In fact, Metchnikoff was forced to follow the chemists’ lead. In his magisterial account, Immunity in Infectious Diseases (1905), he cited the first studies of the biochemical basis of bacterial killing by phagocytes. He noted that following active ingestion, a drop in pH within the digestive vacuoles correlates with bacterial destruction that he thought were enacted by intracellular enzymatic “cytases” ([96], pp. 175–206). And by the time he died a decade later, characterization of “endolysins”—lumped together as unspecified enzymes and bacteriolysins of uncertain origin (i.e., endogenous serum or phagocyte-derived)—were subordinated to the characterizations of soluble serum factors ([97], pp. 296–310). And there matters stood as the focus on acquired immunity—the specificity of the antibody reaction—dominated the first decades of the twentieth century and effectively displaced interest in so-called “natural” immune mechanisms [98, 99].

However, during the closing decades of the twentieth century, following the appreciation of antigen presentation, cytokine regulation, and the various complementary roles of phagocyte-based immunity, the so-called “innate” immune system again received prominent attention. The history of this inflection has not been critically appraised by historians or philosophers of science despite the importance of this chapter in immunology’s development. In this regard, the phagocyte’s respiratory burst would be found at the cross-roads of biochemistry, genetics, cell biology, and immunology to illustrate the mosaic of disciplines required to decipher the mechanisms of host defense and the biochemistry of inflammation [9, 100]. Thus, Metchnikoff’s so-called “vitalism” required elucidation of a dynamic complex system, whose organization and regulation, while still only partially understood, testifies to the prescient insight of his early vision that awaited confirmation by the methodological turn to systems biology and the modern integration of diverse life sciences.