The birth of immunology. II. Metchnikoff and his critics

The presentation of the phagocytic theory of immunity, proposed by Metchnikoff in 1883, was immediately attacked by German pathologists and microbiologists. Led by Baumgarten and Ziegler, criticism was levied against the hypothesis in three general respects: 1) Can an analogy truly be established between leukocyte phagocytosis and feeding of monocellular organisms? 2) What is compelling about the phagocytic process as a universal defensive activity? 3) General philosophic objections were raised, centered upon the accusation of a teleologic formulation. Underlying the argument was the rejection of the notion that the response of phagocytic leukocytes was truly causal in the successful response to infection. We note that the humoral school of immunity was not established until 1888-1890, and the early debate between Metchnikoff and his detractors was not over an alternative theory of an active immune response. There was none. With the development of the humoralist position, in direct response to Metchnikoff's formulation, a true dialogue about immunity, in the modern context of active host response, was initiated. The debate at this point changed to issues of mechanism (cellular versus humoral effectors), and the relative importance of defining innate versus acquired immune processes. Our study traces the scientific and logical basis of the initial rejection of the phagocytosis theory. More broadly, the analysis of this debate elucidates the emergence of a new concept of immunity that rested upon the notion of an active host response. The humoralists erected their theory on Metchnikoff's original scaffold, and the ensuing debate of the nascent science relied on the successful establishment of his basic concept. With the studies of Bordet, Metchnikoff's protégé, the essential resolution of the acrimonious debate was offered. Metchnikoff continued his research by attempting to apply the phagocytosis theory to mechanisms of senility, while immunology oriented itself toward the biochemical definition of immune recognition.     

The birth of immunology: Metchnikoff, the embryologist

Metchnikoff must be viewed first as an embryologist, who, influenced by the Darwinian currents of the 1860s and 1870s, sought to establish a genetic and embryologic unity in phylogeny. His principal early theory that the mesoderm was the origin of endodermal structures enabled him to extend the observation of mesodermal digestive processes to a theory of immunity. Observation of amoeboid phagocytosis was not novel, but Metchnikoff's scientific investigations had prepared him to interpret this activity as a manifestation of a generalized property of the mesoderm. Earlier observers noted the presence of microorganisms and particles in leukocytes, and the notion of phagocytosis had previously been entertained, but only Metchnikoff recognized the importance of phagocytosis in a general scheme of inflammation and to develop an experimental model for its investigation. The observation was thus viewed not solely as an issue of pathology, but rather as a contribution to Metchnikoff's general idea of genetic unity and his hypothesis of a primordial multicellular organism, Parenchymella, later called Phagocytella. It is striking that he ultimately viewed phagocytosis as a question of immunity, considering the context of his research activities, which had been confined to evolution and biology of development. To demonstrate how the famous Messina experiments were extended to a new theory of immunity requires formulating Metchnikoff's recognition of both the importance of phagocytosis for his mesodermal theory and a more general theory of pathology. The result was the genesis of a new idea, immunity.     

Requirement of heat-labile opsonins for maximal phagocytosis of Candida albicans.

The requirement for heat-labile opsonins for phagocytosis of 2 serologically distinct strains and a number of clinical isolates of Candida albicans was examined by a previously described radiometric technique. The results indicated that both strains and all 10 isolates of C. albicans examined required heat-labile opsonins for maximal phagocytosis. The data suggest that heat-labile opsonins play an important role in immunity to C. albicans. However, the possibility that some strains of C. albicans may not utilize heat-labile opsonins cannot be excluded.     

An innately interesting decade of research in immunology

"Nature has provided, in the white corpuscles as you call them-in the phagocytes as we call them-a natural means of devouring and destroying all disease germs. There is at bottom only one genuinely scientific treatment for all diseases, and that is to stimulate the phagocytes." So opined B.B. in G.B. Shaw's The Doctor's Dilemma in a dramatic restatement of a key portion of Ilya Metchnikoff's Nobel Prize address: "Whenever the organism enjoys immunity, the introduction of infectious microbes is followed by the accumulation of mobile cells, of white corpuscles of the blood in particular which absorb the microbes and destroy them. The white corpuscles and the other cells capable of doing this have been designated 'phagocytes,' (i.e., devouring cells) and the whole function that ensures immunity has been given the name of 'phagocytosis'". Based on these insights into the foundation of resistance to infectious disease, Metchnikoff was awarded the 1908 Nobel Prize in Physiology or Medicine together with Paul Ehrlich (Fig. 1). Although both were cited for discoveries in immunity, the contributions of the two men seem worlds apart. Ehrlich's studies did not deal with generic responses to infection, but rather with the highly specific nature of antibodies and their relationship to the cells producing them: "As the cell receptor is obviously preformed, and the artificially produced antitoxin only the consequence, i.e. secondary, one can hardly fail to assume that the antitoxin is nothing else but discharged components of the cell, namely receptors discharged in excess". But biological systems are just that-systems-and the parts need to work together. And so we arrive, a century later, at an appreciation for just how intimately related these two seemingly disparate aspects of host defense really are.     

The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.     

The genius of E. E. metchnikoff— Discoveries over the centuries

On the 15 of May we celebrated the 150th anniversary of the outstanding Russian biologist Elias E. Metchnikoff (1845–1916)—Nobel Prize winner (1908), full and honorary member of many scientific academies of the world. His main works were applied to the zoology of invertebtates, evolution, embryology, immunology, microbiology, infectious pathology, gerontology, etc. Elias Metchnikoff published essays on anthropology, theory of orthobiosis, role of social and social-hygienic factors in solving the problems of old age and life elongation. On 30 May-2 June 1995 an International Symposium dedicated to Metchnikoff's 150th anniversary was held in Moscow. This is a text of the lecture given by us at the opening ceremony.     

Humoral Recognition Factors in the Arthropoda. The Specificity of Chelicerata Serum Lectins

Arthropods have the capacity of recognizing self from non-selfin various defense phenomena including hemolymph clotting, phagocytosis,encapsulation, melanization and clearance of the foreign matterwhich must be initiated by a first step of molecular recognition."Natural" and experimentally induced humoral factors have beendescribed which act as hemagglutinins, bacterial agglutinins,precipitins, bactericidal factors, bacteriolysins, hemolysins,opsonins, clotting factors, and lysozymes. Their exact rolein recognition functions has not been fully explored and theirfunction remains unclear. Among these factors, the carbohydrate-bindingmolecules (lectins) are the best characterized in their specificity,physicochemical properties and molecular structure. Arthropodlectins are multimeric, high molecular weight protein (glycoprotein)molecules with a certain degree of heterogeneity in their specificityand structure. In particular, serum lectins from chelicerates(horseshoe crabs, scorpions and spiders) share a common property:the specificity for sialic acids. Arachnids and merostomes divergedin the earliest Cambrian. Since they occupy markedly differenthabitats, the sialic acid specific lectins most probably area relict rather than an adaptative character. In addition tothis common feature of specificity, lectins from cheliceratesand other arthropods represent heterogeneous populations whichcan bind a wide variety of carbohydrates, many of them presenton bacteria as D-galactose, 2-keto-3-deoxyoctonate, glucuronicacid, N-acetylmuramic acid, and colominic acid. Multiplicityin specificity suggests that serum lectins might contributeas a carbohydrate-based recognition system for the non-self.The requirement for avoiding self recognition would be thatcarbohydrate structures potentially recognized by the systemwould be absent, masked or out of reach of this humoral factoror cell associated recognition factors.     

Surface phagocytosis of Staphylococcus epidermidis and Escherichia coli by human neutrophils: serum requirements for opsonization and chemiluminescence

We examined the serum requirements for surface phagocytosis of Staphylococcus epidermidis and Escherichia coli and for the subsequent chemiluminescent response of human neutrophils. Substantial surface phagocytosis of S. epidermidis occurred in the absence of opsonins, although the presence of 10% pooled or heat-inactivated serum significantly increased phagocytosis. There was no significant difference between these opsonins, indicating that surface phagocytosis of S. epidermidis did not require complement. Unopsonized E. coli were not as readily phagocytized as S. epidermidis (33% versus 57%). In contrast to S. epidermidis optimal phagocytosis of E. coli required complement as 10% heat inactivated donor serum (HHS) was significantly less effective as an opsonin than 10% pooled healthy donor serum (PHS). The time kinetics for phagocytosis of each organism were similar, with most of the phagocytosis occurring in the first 10 min. The chemiluminescent response of neutrophils produced discrepant results. Maximal chemiluminescence was observed when neutrophils were stimulated with bacteria opsonized in PHS. The response to HHS-opsonized bacteria was less, and chemiluminescence to unopsonized bacteria was only marginally higher than the control, even though there was relatively good phagocytosis. These results define the opsonic requirements for surface phagocytosis of S. epidermidis and E. coli and indicate that although complement may not be required for phagocytosis, it is necessary for generation of a maximal oxidative burst, and thus may be essential for efficient intracellular killing.     

Surface phagocytosis of Staphylococcus epidermidis and Escherichia coli by human neutrophils: serum requirements for opsonization and chemiluminescence

Abstract We examined the serum requirements for surface phagocytosis of Staphylococcus epidermidis and Eschericia coli and for the subsequent chemiluminescent response of human neutrophils. Substantial surface phagocytosis of S. epidermidis occured in the absence of opsonins, although the presence of 10% pooled or heat-inactivated serum significantly increased phagocytosis. There was no significant difference between these opsonins, indicating that surface phagocytosis of S. epidermidis did not require complement, Unopsonized E. coli were not as readily phagocytized as S. epidermidis (33% versus 57%). In contrast to S. epidermidis optimal phagocytosis of E. coli required complement as 10% heat inactivated donor serum (HHS) was significantly less effective as an opsonin than 10% pooled healthy donor serum (PHS). The time kinetics for phagocytosis of each organism were similar, with most of the phagocytosis iluminescent response of neutrophils produced discrepant results. Maximal chemiluminescence was observed when neutrophils were stimulated with bacteria opsonized in PHS. The response to HHS-opsonized bacteria was less, and chemiluminescence to unopsonized bacteria was only marginally higher than the control, even though there was relatively good phagocytosis. These results define the opsonic requirements for surface phagocytosis of S. epidermidis and E. coli and indicate that although complement may not be required for phagocytosis, it is necessary for generation of a maximal oxidative burst, and thus may be essential for efficient intracellular killing.     

Agglutinin-mediated phagocytosis-associated generation of superoxide anion and nitric oxide by the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii

Hemocyte mediated phagocytosis is one of the vital components of innate defence mechanisms in crustaceans and this phagocytic process is aided by serum agglutinins. However, literature on agglutinin mediated opsono-phagocytosis is unclear in the case of Macrobrachium rosenbergii hemocytes. Further, very few studies in the case of superoxide anion generation and none with regard to nitric oxide generation during phagocytosis exist among crustaceans. We investigated the occurrence of agglutinins in the serum and the role of serum agglutinins in mediating phagocytosis by the hemocytes. We show that the prawn serum possesses agglutinins that function as opsonins during phagocytosis of HB RBC by the hemocytes. Hemagglutination-inhibition assays revealed the specificity of serum agglutinins for N-acetylated hexoses, namely GalNAc, GlcNAc and ManNAc, with a higher affinity for ManNAc. In addition, ManNAc was able to inhibit the phagocytic response (by about 60%) of the hemocytes against serum pretreated HB RBC, wherein the serum was previously treated with ManNAc. We next investigated the ability of the hemocytes to generate superoxide anion and nitric oxide during HB RBC phagocytosis and results show generation of both these free radicals. In addition, there was an enhancement in generation (75% increase) of these free radicals during agglutinin mediated opsonophagocytosis, when compared to buffer treated targets and interestingly this enhanced generation was inhibited by ManNAc (27% for superoxide anion and 36% for nitric oxide), an inhibitory sugar for phagocytosis. Inhibition of phagocytosis induced superoxide anion generation by DPI (53%), sodium azide (56%) and tropolone (61%), reveals the possible involvement of NADPH-oxidases, peroxidases and probably phenoloxidases, respectively, in the generation of superoxide anion. Similarly, decrease in nitric oxide generation in the presence of l-NIO (47%) during phagocytosis lends support to the role of nitric oxide generation during cellular immune processes. These findings thus suggest a role for superoxide anion and nitric oxide in the innate defense mechanism, namely phagocytosis, in Macrobrachium rosenbergii.     

Who was... Emil von Behring?

Emil von Behring was first in the line of distinguished immunologists to win the Nobel Prize for Physiology and Medicine. His contributions to our knowledge of immunity ignited an impassioned argument between French and German scientists at the end of the 19th century, the first of many scientific debates in the immunological world.     

The Nobel Prize as a Reward Mechanism in the Genomics Era: Anonymous Researchers,Visible Managers and the Ethics of Excellence

The Human Genome Project (HGP) is regarded by many as one of the major scientific achievements in recent science history, a large-scale endeavour that is changing the way in which biomedical research is done and expected, moreover, to yield considerable benefit for society. Thus, since the completion of the human genome sequencing effort, a debate has emerged over the question whether this effort merits to be awarded a Nobel Prize and if so, who should be the one(s) to receive it, as (according to current procedures) no more than three individuals can be selected. In this article, the HGP is taken as a case study to consider the ethical question to what extent it is still possible, in an era of big science, of large-scale consortia and global team work, to acknowledge and reward individual contributions to important breakthroughs in biomedical fields. Is it still viable to single out individuals for their decisive contributions in order to reward them in a fair and convincing way? Whereas the concept of the Nobel prize as such seems to reflect an archetypical view of scientists as solitary researchers who, at a certain point in their careers, make their one decisive discovery, this vision has proven to be problematic from the very outset. Already during the first decade of the Nobel era, Ivan Pavlov was denied the Prize several times before finally receiving it, on the basis of the argument that he had been active as a research manager (a designer and supervisor of research projects) rather than as a researcher himself. The question then is whether, in the case of the HGP, a research effort that involved the contributions of hundreds or even thousands of researchers worldwide, it is still possible to “individualise” the Prize? The “HGP Nobel Prize problem” is regarded as an exemplary issue in current research ethics, highlighting a number of quandaries and trends involved in contemporary life science research practices more broadly.     

Brain plasticity and mental processes: Cajal again

The year 2006 marks the 100th anniversary of the first Nobel Prize for Physiology or Medicine for studies in the field of the Neurosciences jointly awarded to Camillo Golgi and Santiago Ramón y Cajal for their key contributions to the study of the nervous system. This award represented the beginning of the modern era of neuroscience. Using the Golgi method, Cajal made fundamental, but often unappreciated, contributions to the study of the relationship between brain plasticity and mental processes. Here, I focus on some of these early experiments and how they continue to influence studies of brain plasticity.     

Phagocytosis by bovine polymorphs of glass-adherent Escherichia coli in the presence and absence of serum

The influence of serum on the phagocytosis and killing of Escherichia coli adherent to glass (‘surface phagocytosis’) was investigated by using 2 strains known to require opsonisation when phagocytosis takes place in suspension. Although phagocytosis of the unencapsulated strain occurred in the absence of serum, serum was necessary for the uptake of the encapsulated strain, and improved the uptake of both strains. Killing of ingested bacteria appeared to be independent of serum. Antibodies were not an absolute requirement for the promoting effect of serum, and the necessity for complement (Cp) appeared to depend on the presence of antibodies and on the strain tested. The results suggest that serum components other than opsonins (antibodies and Cp) contributed to the serum-enhanced uptake of bacteria.     

Signature miRNAs involved in the innate immunity of invertebrates

The innate immune system, including the cell-based immunity (mainly apoptosis and phagocytosis) and the humoral immunity (such as pro-phenoloxidase system), is the first defense line of animals against the infection of pathogens in a non-specific manner, which is fine regulated through the gene expression regulations. The microRNAs (miRNAs) are recognized as important regulators of gene expression. To date, however, a comprehensive view about the regulation of innate immunity by miRNAs is not available. To address this issue, the signature miRNAs involved in the innate immunity were characterized in this study. The phagocytosis, apoptosis and phenoloxidase (PO), a key enzyme in the pro-phenoloxidase system, of invertebrate shrimp were activated or inhibited, followed by the small RNA sequencing. The results showed that a total of 24 miRNAs took great effects on phagocytosis, apoptosis or the pro-phenoloxidase system, which were further confirmed by Northern blots. Among the 24 innate immunity-associated miRNAs, 21 miRNAs were conserved in animals, suggesting that these miRNAs might share the similar or the same functions in different species of animals. Based on degradome sequencing and prediction of target genes, it was found that the miRNAs might mediate the regulations of phagocytosis, apoptosis or pro-phenoloxidase system by targeting different genes. Therefore our study presented the first comprehensive view of the miRNAs associated with innate immunity, which would facilitate to reveal the molecular events in the regulation of innate immunity.     

Interaction of Candida albicans with neutrophils: effect of phenotypic changes in yeast cell-surface composition

The susceptibility of four strains of Candida albicans to phagocytosis and intracellular killing by rabbit peritoneal neutrophils was investigated. Two of the strains, isolated from active infections, were known to synthesize a surface layer of mannoprotein fibrils in response to growth on 500 mm-galactose; the other strains, from asymptomatic carriers, lacked this capability. The presence of serum opsonins greatly enhanced phagocytosis of all four strains and, following opsonization, phagocytosis of an infective strain was equally rapid after growth on either 500 mm-galactose or 50 mm-glucose. In the absence of opsonins, galactose-grown infective strains were phagocytosed faster than either glucose-grown infective strains or galactose-grown carrier strains. These differences in phagocytic uptake were paralleled by differences in neutrophil chemiluminescence response. Intracellular killing of galactose-grown infective strains was only half that of glucose-grown infective strains or galactose-grown carrier strains after incubation for 60 min. Pretreatment of neutrophils with extracellular polymeric material, which contains the surface fibrils, completely inhibited intracellular killing. These results indicate that production of the fibrillar layer promotes yeast virulence by increasing resistance to intracellular killing, although it may enhance phagocytosis in locations where opsonic activity is poor.     

Role of serum factors in the phagocytosis of weakly or heavily encapsulated Cryptococcus neoformans strains by guinea pig peripheral blood leukocytes

We investigated the opsonic activity of the serum factors affecting phagocytosis of Cryptococcus neoformans in vitro to elucidate the role of humoral factors in the host defense mechanisms against cryptococcosis. Two strains of C. neoformans, one heavily and one weakly encapsulated, were used. Guinea pig peripheral blood leukocytes (PBLs) were used for phagocytosis. The viable weakly encapsulated cells were ingested effectively by PBLs, in the presence of guinea pig normal fresh serum, while the heavily encapsulated cells were not ingested. Neither immune serum, its IgG fraction alone, nor heated serum promoted the phagocytosis of either the weakly or heavily encapsulated strain. On the other hand, immune serum promoted adherence of PBLs to viable cells of the heavily encapsulated strain, forming rosettes in the presence of fresh serum. A substantial amount of C3b component was detected on yeast cells when weakly encapsulated cells were incubated with human fresh serum, or heavily encapsulated cells were incubated with rabbit immune serum together with human fresh serum. Serum chelation experiments also indicated that the factors involved in the alternative complement pathway are opsonins for the weakly encapsulated strain. These results suggest that the alternative pathway plays an important normal opsonic role for weakly encapsulated strains and that specific antibody plays an immune opsonic role for heavily encapsulated strains of C. neoformans via the classical pathway of complement activation.     

Recent updates in cancer immunotherapy: a comprehensive review and perspective of the 2018 China Cancer Immunotherapy Workshop in Beijing

The immune system is the hard-wired host defense mechanism against pathogens as well as cancer. Five years ago, we pondered the question if the era of cancer immunotherapy was upon us (Li et al., Exp Hem Oncol 2013). Exciting progresses have been made at all fronts since then, including (1) sweeping approval of six agents by the US Food and Drug Administration (FDA) to block the PD-1/PD-L1 pathway for treatment of 13 cancer types; (2) a paradigm shifting indication of PD-1 and CTLA4 blockers for the management of a broad class of cancers with DNA mismatch repair defect, the first-ever tissue agnostic approval of cancer drugs; (3) real world practice of adoptive T cell therapy with two CD19-directed chimeric antigen receptor T cell products (CAR-T) for relapsed and/or refractory B cell malignancies including acute lymphoid leukemia and diffuse large B cell lymphoma, signaling the birth of a field now known as synthetic immunology; (4) the award of 2018 Nobel Prize in Physiology and Medicine from the Nobel Committee to Tasuku Honjo and James Allison “for their discovery of cancer medicine by inhibition of negative immune regulation” (www.nobelprize.org/prizes/medicine/2018); and (5) the emerging new concept of normalizing rather than amplifying anti-tumor immunity for guiding the next wave of revolution in the field of immuno-oncology (IO) (Sanmamed and Chen, Cell 2018).This article will highlight the significant developments of immune-oncology as of October 2018. The US FDA approved indications of all seven immune checkpoint blockers, and two CD19-directed CAR-T products are tabulated for easy references. We organized our discussion into the following sections: introduction, cell therapy, emerging immunotherapeutic strategies, expediting oncology drug development in an era of breakthrough therapies, new concepts in cancer immunology and immunotherapy, and concluding remarks. Many of these topics were covered by the 2018 China Cancer Immunotherapy Workshop in Beijing, the fourth annual conference co-organized by the Chinese American Hematologist and Oncologist Network (CAHON), China FDA (CFDA; now known as China National Medical Product Administration (NMPA)), and the Tsinghua University. We significantly expanded our discussion of important IO developments beyond what were covered in the conference, and proposed a new Three Rs conceptual framework for cancer immunotherapy, which is to reverse tolerance, rejuvenate the immune system, and restore immune homeostasis. We conclude that the future of immuno-oncology as a distinct discipline of cancer medicine has arrived.     

Phosphatidylserine recognition by phagocytes: a view to a kill

The redistribution of phosphatidylserine (PS) to the external surface of the plasma membrane is a key element of apoptotic cell recognition and is a molecular cue that dying cells should be engulfed. Phagocytes interact with PS on apoptotic cells through either the PS receptor or secreted bridging proteins called opsonins. The study of two secreted PS opsonins, MFG-E8 and Gas6 and their receptors alphavbeta5 (and alphavbeta3) integrin and Mer tyrosine kinase, respectively, have provided insights into the temporal and spatial aspects of Rac1 activation following the recognition and internalization of apoptotic cells. Disruption of PS opsonins and their signaling pathways often manifest conditions of inflammation and autoimmune disease. Here, we review recent studies involving PS opsonins, their receptors and their role in the phagocytosis of apoptotic cells.