Virchows Arch (2015) 466:111–116 DOI 10.1007/s00428-014-1677-4

ORIGINAL ARTICLE

The contribution of Paul Ehrlich to histochemistry: a tribute on the occasion of the centenary of his death Igor Buchwalow & Werner Boecker & Markus Tiemann

Received: 26 September 2014 / Revised: 20 October 2014 / Accepted: 22 October 2014 / Published online: 31 October 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Paul Ehrlich is the founder of a number of areas in biomedical research: in the first line, immunology and chemotherapy. Aim of this historical note to the centenary of Paul Ehrlich’s death is to commemorate his tribute to the establishment and development of histochemistry. Keywords Paul Ehrlich . Pathfinder in histochemistry . Histology . Hematology . Chemotherapy . Immunology

Paul Ehrlich (14 March 1854–20 August 1915) (Fig. 1) is the founder of a number of areas in biomedical research. On the basis of his research career as a histologist, hematologist, immunologist, organic chemist, or pharmacologist, Ehrlich can be rightfully recognized as giant among giants. There are numerous reviews [1, 2, 5, 26, 27, 29, 35], books [3, 31, 33, 36, 37], and films [8] portraying his life and his tribute to immunology and chemotherapy. The aim of this historical note to the centenary of his death is to commemorate his role in the establishment and development of histochemistry. Born in Strehlen near Breslau (the German Province of Silesia, now in Poland), Paul Ehrlich was the second child of Ismar and Rosa Ehrlich. Ismar Ehrlich, the leader of the local Jewish community, gave his first son the Christian name Paul. As a schoolboy, Paul was inspired by his mother’s cousin, the renowned pathologist Karl Weigert (1845–1904). Karl Weigert was the first who introduced aniline dyes in histology and bacterial diagnostics and owned one of the first microtomes. He was only 9 years older than Paul, and the two became close friends. Paul was fascinated by the process of staining tissue substances for microscopic examination and retained that interest during his subsequent medical studies. I. Buchwalow (*) : W. Boecker : M. Tiemann Institute for Hematopathology, Fangdieckstr. 75a, 22547 Hamburg, Germany e-mail: [email protected]

Ehrlich studied medicine at the Universities of Breslau, Strassburg, Freiburg-im-Breisgau, and Leipzig between 1872 and 1877. Continuing the interest, instilled by his cousin, in staining tissues for microscopic study, Ehrlich spent his eighth university semester in Freiburg im Breisgau investigating the red dye dahlia (monophenylrosanilin). This gave rise to his first publication Beiträge zur Kenntnis der A n i l i n f ä r b u n g e n u n d i h re r Ve r w e n d u n g i n d e r mikroskopischen Technik (Contributions to the applications of aniline dyes in microscopic technique, 1877) [9] (Fig. 2). It was the first successful application of rosanilin stain for histological staining. Remarkably, another derivative of rosanilin—triphenylrosanilin, or aniline blue—was later found suitable for staining collagen in Masson’s trichrome and Mallory’s methods for connective tissue. Ehrlich was not an outstanding student. He was obsessed with tissue staining, as one of his professors recalled when he introduced Ehrlich to Robert Koch. “That is little Ehrlich. He is very good at staining, but he will never pass his examinations” [31, 36]. However, in 1878, Ehrlich brilliantly obtained at the Leipzig University a doctorate with a dissertation entitled Beiträge zur Theorie und Praxis der Histologischen Färbung (Contributions to the Theory and Practice of Histological Staining, Thesis, Leipzig University, 1878) [10]. One of the most outstanding results of his dissertation investigations was the discovery of a new cell type that he named Mastzellen (“mast cells” from the German word for an animal-fattening feed, Mast) [7]. At that time, this focus on chemistry was unusual for a medical dissertation. In his dissertation, Ehrlich presented the entire spectrum of known staining techniques with the in-depth explication of the chemistry of the dyestuffs employed, especially aniline dyes. The term aniline ascends toward an indigo-yielding plant, Añil (Indigofera suffruticosa). This term was introduced in 1841 by Carl Julius Fritzsche (Adjunct of the St. Petersburg Academy of Sciences, Russia), who treated indigo with

112

Fig. 1 Paul Ehrlich

caustic potash and obtained an oil that he named aniline. A year later, a Russian organic chemist Nikolay Zinin (University of Kazan, Russia) performed chemical synthesis of aniline

Virchows Arch (2015) 466:111–116

via conversion of nitro aromates like nitrobenzene to amines by reduction with sodium sulfides (Zinin reaction) (Fig. 3). In 1856, an Englander William Henry Perkin discovered that aniline could be used to make intense coloring agents. The subsequent ascendancy of the German dyestuff industry over British manufacturers had led to the burst of commercial production of synthetic dyes in Germany and to the following introduction of aniline dyes into medicine. The interest in aniline dyes accompanied Ehrlich throughout his career starting from the application of rosanilin stain for histological staining while he was still a student [9] and running until the first decades of the 20th century when he created his famous arsphenamine (compound 606, or Salvarsan), that also had its roots in the aniline chemistry (Fig. 4). Salvarsan was the first successful chemotherapy agent [19] foregoing Gerhard Domagk’s sulfanilamides and Alexander Fleming’s penicillin. Gerhard Domagk’s [34] sulphonamides were the best proof that Ehrlich was right: dyes could act as antibacterial agents.

Fig. 2 Two first pages from the first publication of Paul Ehrlich (1877) Beiträge zur Kenntniss der Anilinfärbungen und ihrer Verwendung in der mikroskopischen Technik. Archiv f. mikrosk. Anat. 13:263–277

Virchows Arch (2015) 466:111–116

113

Fig. 3 The reduction of nitrobenzene to aniline was first performed by a Russian organic chemist Nikolay Zinin in 1842 using inorganic sulfide as a reductant (Zinin reaction)

After obtaining his doctorate in 1878, Ehrlich was appointed as an assistant to Professor Frerichs at the Berlin Medical Clinic (Charité), who gave him every facility to continue the work with his dyes to stain tissues. Exploring the avidity of cells and tissues for certain dyes and using dyes classified as being basic, acid, and neutral for the staining of granules in blood cells, Ehrlich made it possible to distinguish between basophils, neutrophils, and eosinophils, as well as between lymphocytes, plasma cells, and mast cells. This led to the capability to diagnose numerous blood diseases [17, 24]. With his investigation of white blood cells, he had created the basis for systematic classification of leukemias. Ehrlich also laid the basis for the analysis of anemia, as he discovered the precursors of erythrocytes and demonstrated the existence of nucleated red blood cells, which he subdivided into normoblasts, megaloblasts, microblasts, and poikiloblasts [20, 21].

Fig. 4 Three entries in Paul Ehrlich’s laboratory notebook dated 1906– 1912 illustrate the development of Salvarsan 606

Ehrlich’s duties at the Charité included also analyzing patient blood and urine specimens. His experimental work at the Charité resulted in numerous diagnostic methods [13, 18, 22, 23], some of which still remain in use. For example, with the diazo reaction (Ehrlich reagent), he was the first to detect the presence of bilirubin in the urine of patients with jaundice [12]. In 1882, Robert Koch announced to the Berlin Society for Physiology his discovery of the causal agent of tuberculosis. Ehrlich later described this lecture as his “greatest experience in science”. In the same year, Ehrlich published his method of staining mycobacteria. He extended comparable staining methods to bacteria and protozoa and rendered Koch’s discovery of the tubercle bacillus immediately more important by showing that its failure to stain in aqueous dye solutions could be circumvented by use of basic dyes in an aqueous-aniline oil solution, which penetrated the bacillary coating and then remained acid-fast [11]. This result was honored by Koch and medical circles as a highly valuable contribution to diagnostics. This method served as a basis for the subsequent modifications introduced by Ziehl and Neelsen, which are still used today [4]. A derivative of this technique is the Gram method of staining bacteria, so much used by bacteriologists until today. In 1885, a remarkable monograph, Das Sauerstoffbedürfnis des Organismus (The need of the organism for oxygen) [14], reporting his investigations into the distribution of oxygen in animal tissues and organs, gained widespread attention from medical scientists. Challenging the assertion of the renowned German physiologist Eduard Pflüger (1829–1910) that tissue oxidation and reduction entail direct entry and exit of oxygen, Ehrlich contended that these processes involve withdrawal and insertion of hydrogen atoms. This concept he splendidly demonstrated with his famous enzymatic NADI reaction—the oxidative reaction between άnaphtol and dimethyl-p-phenylendiamine resulting in formation of indophenol blue dye (Fig. 5). The enzyme responsible was originally called indophenol oxidase. Today, the NADI reaction carried out on formaldehyde-fixed and paraffin-embedded preparations remains still in use for demonstration of myeloperoxidase in histopathology and hematology for the differential diagnosis of hematological disorders.

114

Virchows Arch (2015) 466:111–116

Fig. 5 NADI Reaction: Dimethyl-p-phenylendiamine reacts with the coupler (ά-Naphthol) to a colorless leuco compound. By oxidation of the leuco compound, a water-soluble indophenol dye is formed (naphthol blue). Adapted from

At the end of the 19th century, cytochrome oxidase was unknown. It was decades later, that Keilin and Hartree [28] showed that indophenol oxidase, Atmungsferment, and cytochrome oxidase were one and the same enzyme, and that the NADI reaction in effect demonstrated the enzymatic activity of the cytochrome oxidase catalyzing the oxidation of its substrate—reduced cytochrome C (ferrocytochrome C)—to ferricytochrome C in the presence of oxygen. It is worth noting that Erlich’s indophenol oxidase reaction underlies the main principle of enzyme histochemical demonstration of oxidoreductases, enzymes that catalyze the reactions of biological oxidation associated with electron transfer from one molecule (donor of electrons) to another (acceptor of electrons). In the following decades, other chromogens were introduced into enzyme histochemistry of oxidoreductases. For routine practice, one of the most important chromogens is diaminobenzidine (DAB). Like the components of the NADI reaction, DAB (also a derivative of aniline) serves as a donor of electrons in catalyzed oxidation-reduction reactions. In the modern enzyme histochemistry, DAB is primarily used for demonstration of various oxidoreductases—in the first line peroxidase. Visualization of antibodies labeled with peroxidase is nowadays broadly used in immunohistochemistry [6]. The NADI reaction with a full right may be regarded as a milestone in the establishment of enzyme histochemistry [30]—a completely new dimension in those days. Notably, the NADI reaction not only permitted the first use of the principle of electron transfer from one molecule to another resulting in formation of a colored product, but also the first use of ά-naphtol as a coupling agent for azo dye formation in enzymatic reactions. In the following decades, other naphtol derivatives were introduced into enzyme histochemistry as substrates in the azo-coupling reaction for demonstration

of other classes of enzymes such as phosphatases and peptidases. Ehrlich’s great achievements in an entirely new field of study interrelating chemistry, biology, and medicine appeared in those days; however, as a source of problems during his further career. Much of his work was rejected by the medical profession, which lacked the necessary chemical knowledge. The reason for his conflict was his steadily rising interest in research for dyes and marking agents with selective staining properties. It was alleged that his only interest was the “affinity” of the stains to what they stained and nothing else. Some of his colleagues whispered to his address that he should give up his “pipe” dreams of discoveries through chemicals [31, 36]. Finally, it meant that there was no suitable professorship in sight for Ehrlich. When the Ehrlich’s principal at the Charité, Professor Frerichs, died suddenly in 1885, and the more conservative Karl Gerhardt succeeded him, Ehrlich found his research disturbingly impeded. After mid-1889 without appointment, Ehrlich set up a small private laboratory in a rented flat and launched a series of fundamental studies in immunity, with which his name will always be associated. After describing his studies in this field in two papers entitled Experimentelle Untersuchungen über Immunität (Experimental Studies on Immunity, Part 1 and Part 2, 1891) [15, 16], he continued his immunological studies in a small laboratory at the newly founded Institute for Infectious Diseases in Berlin, of which Koch had become director. Ehrlich worked here for more than 3 years without salary, despite his appointment as extraordinary professor at Berlin University in 1891. Around that time, Emil Behring (1854–1917) had worked in this institute on developing an antiserum for treating diphtheria and tetanus, but with inconsistent results. Koch suggested that Behring and Ehrlich should cooperate on the

Virchows Arch (2015) 466:111–116

project. This joint work was successful to the extent that Ehrlich was quickly able to increase the level of immunity of the laboratory animals based on his experience with mice. Ehrlich conceived a method for standardizing therapeutic serums. This method is still in use today. Clinical tests with diphtheria serum early in 1894 were successful; and in August, the chemical company Hoechst started to market “Diphtheria Remedy synthesized by Behring-Ehrlich”. Later however, Behring outfoxed Ehrlich in a commercial arrangement with the Hoechst Company and garnered for himself Ehrlich’s share of the considerable profits from the sale of antibodies against diphtheria toxin. In addition, Behring on his part schemed against Ehrlich at the Prussian Ministry of Culture and from 1900 Ehrlich refused to collaborate with him. Another by-product of Ehrlich’s ingenuity dates back to the mid-1889, the time when he was still unemployed and worked in his small private laboratory. There, he continued his research on methylene blue for histological staining. Ehrlich obtained methylene blue from the company Meister Lucius & Brüning AG (later renamed Hoechst AG) and engaged in a long collaboration with this company. Ehrlich found that methylene blue stained the long appendages of nerve cells, the axons. It was the opinion of the influential German anatomist and neurologist Ludwig Edinger (1855–1918) that Ehrlich thus had opened up a major new topic in the field of neurology. Around the same time, Ehrlich continued his work in his private laboratory on in vivo staining of bacteria with methylene blue. Since the parasite family of Plasmodiidae—which includes the malaria pathogen—could be selectively stained with methylene blue, he got the idea that it might be used therapeutically as treatment for malaria [25]. Interest in the use of methylene blue as an antimalarial had revived [32], especially due to its low price. Therapeutic progress coming from dye-chemistry following the use of methylene blue gave birth to various families of chemotherapeutics: antiseptics, antiparasitic, and antibacterial drugs [34]. Ehrlich reasoned that if a compound could be made that selectively targeted a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity. Hence, magische Kugel (“magic bullet”, his term for an ideal therapeutic agent) would be created that killed only the targeted organism. The Ehrlich’s concept of a “magic bullet” was to some extent realized decades later by the invention of monoclonal antibodies, as they bring the corresponding toxin to the target (bacterial agent or cancer cell) and provide a very specific binding affinity. Notably, the mechanisms of antigen-antibody binding were also first elucidated by Paul Ehrlich, and the term antikörper (the German word for “antibody”) was first used in a text by Paul Ehrlich in the conclusion of his articles “Experimental Studies on Immunity” [15, 16], published in October 1891. Despite the varied nature of his investigations, a unifying principle is discernible throughout. Ehrlich’s work with what

115

he called his “dye-cupboard” laid the foundation for his thinking about both immunity and chemotherapy. One can guess that Ehrlich’s concept of a “magic bullet” rose from his early studies on dyes and from his observations that aniline dyes specifically target the bacterial pathogen which they can exterminate. His later observations, that antibodies can destroy bacteria without harming the surrounding tissue, apparently served to further reinforce his ideas about the “magic bullet”. Ehrlich was no stranger to controversy. He had a longrunning, often bitter, disagreement with Elie Metchnikoff, discoverer of the macrophage, about the primacy of antibodies (Ehrlich) or phagocytes (Metchnikoff) in immunity. Ironically, the 1908 Nobel Prize was awarded to both men. After sharing the 1908 Nobel Prize with Metchnikoff, awarded in recognition of their work on immunity, Ehrlich was further renominated in 1912 and 1913 for his contributions to chemotherapy; but before the question was settled, Ehrlich had died on August 20, 1915. His friends remembered Paul Ehrlich as enthusiastic and good-humored, at times even bantering. His tastes in general literature aspired no higher than Conan Doyle, he lacked a feeling for art and was refreshed by simple music. His health was not robust, he ate little and smoked heavily; but at work, he was energetic, enthusiastic, and bold. He possessed technical ingenuity, virtuosity in “test-tube” chemistry, and the faculty of three-dimensionally visualizing benzene rings and structural chemical formulas. His perfectionism and a phenomenal capacity for deduction were combined with anarchistic tendencies and with the unique capacity to direct several lines of research simultaneously. This combination gave rise to a personality that some have ventured to describe as that of a genius [5, 31, 36]. On the centenary of Paul Ehrlich’s death, this German researcher deserves to be remembered as a pioneer in a large number of scientific disciplines. Despite the scope of his many achievements in different disciplines, Paul Ehrlich’s name will undoubtedly remain linked to immunology and to the birth of chemotherapy. However, the fundamental contributions to the development of histochemistry of this brilliant scientist in the biomedical field remain an equally essential element of his legacy. Conflict of interest We declare that we have no conflict of interest.

References 1. Albert N (1915) Paul Ehrlich. Arch Dermatol Syph 121:557–578 2. Apolant H (1914) Paul Ehrlich: eine Darstellung seines wissenschaftlichen Wirkens. G. Fischer, Jena 3. Baumler E (1984) Paul Ehrlich: scientist for life. Holmes & Meier Pub, Germany 4. Bishop PJ, Neumann G (1970) The history of the Ziehl-Neelsen stain. Tubercle 51:196–206

116 5. Bosch F, Rosich L (2008) The contributions of Paul Ehrlich to pharmacology: a tribute on the occasion of the centenary of his Nobel Prize. Pharmacology 82:171–179 6. Buchwalow IB, Boecker W (2010) Immunohistochemistry: basics and methods. Springer Heidelberg, Dordrecht 7. Crivellato E, Ca B, Mallardi F et al (2003) Paul Ehrlich’s doctoral thesis: a milestone in the study of mast cells. Br J Haematol 123:19–21 8. Dieterle W (1940) Film, Dr. Ehrlich’s magic bullet. USA 9. Ehrlich P (1877) Beiträge zur Kenntniss der Anilinfärbungen und ihrer Verwendung in der mikroskopischen Technik. Arch Mikrosk Anat 13:263–277 10. Ehrlich P (1878) Beiträge für Theorie und Praxis der histologischen Färbung. vol Doktor. Leipzig University, Leipzig, p 65 11. Ehrlich P (1882) Über die Färbung der Tuberkelbazillen. Deutsche medizinische Wochenschrift:35–37 12. Ehrlich P (1883) Verhalten des Harns zu Sulfodiazobenzol. Z Anal Chem 22:301–301 13. Ehrlich P (1884) Sulfodiazobenzol als Reagens auf Bilirubin. Z Anal Chem 23:275–276 14. Ehrlich P (1885) Das Sauerstoff-Bedürfniss des Organismus. Eine farbenanalytische Studie. Habilitationsschrift. vol Habilitation. Universität Berlin, Berlin 15. Ehrlich P (1891) Experimentelle Untersuchungen über Immunität. I. Ueber Ricin. Dtsch Med Wochenschr 17:976–979 16. Ehrlich P (1891) Experimentelle Untersuchungen über Immunität. II. Ueber Abrin. Dtsch Med Wochenschr 17:1218–1219 17. Ehrlich P (1891) Farbenanalytische Untersuchungen zur Histologie und Klinik des Blutes. A. Hirschwald, Berlin 18. Ehrlich P (1893) Konstitution, Verteilung und Wirkung chemischer Koerper. Aeltere und neuere Arbeiten. G. Thieme, Leipzig 19. Ehrlich P (1909) Beiträge zur experimentellen Pathologie und Chemotherapie. Akademische Verlagsgesellschaft, Leipzig 20. Ehrlich P (1910) Anæmia by Geh. Obermedizinalrat Professor Dr. P. Ehrlich. … . and Dr. A. Lazarus. Part I. Volume I. Normal and pathological histology of the blood. Second edition (enlarged and to a great extent rewritten). Rebman Company, New York

Virchows Arch (2015) 466:111–116 21. Ehrlich P, Lazarus A (1898) Die Anaemie. A. Hölder, Wien 22. Ehrlich P, Penzoldt F (1883) Ueber die Anwendung der Diazobenzolsulfosäure zum Nachweis von Traubenzucker. Z Anal Chem 22:466–467 23. Ehrlich P, Pröscher F (1901) Dimethylamidobenzaldehydreaction. Z Anal Chem 40:193–193 24. Ehrlich P, Woodhead GS, Myers W et al (1900) Histology of the blood, normal and pathological. University Press, Cambridge 25. Guttmann P, Ehrlich P (1891) Ueber die Wirkung des Methylenblau bei Malaria. Berliner Klin Wochenschr 39:953–956 26. Kasten FH (1996) Paul Ehrlich: pathfinder in cell biology. 1. Chronicle of his life and accomplishments in immunology, cancer research, and chemotherapy. Biotech Histochem Off Publ Biol Stain Comm 71:2–37 27. Kaufmann SH (2008) Paul Ehrlich: founder of chemotherapy. Nat Rev Drug Discov 7:373 28. Keilin D, Hartree EF (1938) Cytochrome oxidase. Proc R Soc Lond B 125:171–186 29. Liebenau J (1990) Paul Ehrlich as a commercial scientist and research administrator. Med Hist 34:65–78 30. Lojda Z, Gossrau R, Schiebler T (1976) Enzyme histochemistry. A laboratory manual. Springer, Berlin 31. Mann J (1999) The elusive magic bullet: the search for the perfect drug. Oxford University Press, Oxford 32. Meissner PE, Mandi G, Coulibaly B et al (2006) Methylene blue for malaria in Africa: results from a dose-finding study in combination with chloroquine. Malar J 5:84 33. Metchnikoff E (1939) The founders of modern medicine; Pasteur, Koch, Lister. Walden Publication, New York 34. Otten H (1986) Domagk and the development of the sulphonamides. J Antimicrob Chemother 17:689–690 35. Prull CR (2003) Part of a scientific master plan? Paul Ehrlich and the origins of his receptor concept. Med Hist 47:332– 356 36. Silverman M (1948) Magic in a bottle. Macmillan Co, New York 37. Silverstein A (2001) Paul Ehrlich’s receptor immunology: the magnificent obsession. Academic, New York