Connective Tissue Research and the Rheumatic Diseases

By WALTER BA~ER

HE THEME of this lecture series, “Contributions of Clinical Medicine T to Fundamental Science,” has been abundantly documented by previous speakers. Although the exact sequence of events which began with a clinical observation and ended with an advance in basic science was not always apparent, it was clearly evident that the interplay between the clinic and the laboratory is freely reversible and productive of feedback mechanisms which are of great importance.

Frequently, clinical problems provide the stimuIus for extensive investiga- tion in areas which might not otherwise receive much attention. In these instances, “disease orientation” can enhance basic research in medical biology. In this setting, the relationship between clinical medicine and basic science is a closer one. The vital link between clinician and scientist may be the constant interchange of ideas or a planned cooperative endeavor. In either instance, the more intimate the association between the two, the more often will this type of link be forged. The advantages of this association are many. Its success requires mutual respect, understanding and belief in the intrinsic value of creating knowledge.

Certain of the speakers have cited specific instances in which clinical ob- servations have led to advance of a purely basic and fundamental nature. One famous example is the well documented study of Alexis St. Martin by Dr. Beaumont, conducted in my former homeland, Northern Michigan. Beaumont, working alone at Fort Crawford, made admirably careful observations on his world-famous patient. He had full faith in the importance of his scientific work and of its permanent value. Beaumont’s classic experiments on digestion are one of America’s gifts to science.

Drs. Bearn and Pauling told us of some of the exciting recent developments arising from biochemical studies of some of the genetically based diseases of metabolism. These studies have led to specific observations of considerable fundamental, as well as practical, importance.

I wish to explore today some of the problems pertaining to a group of wide- spread, chronic crippling diseases of unknown etiology and pathogenesis.

From the Medical Clfnlc of the Massachusetts General Hospitol and the Department of Medicine, Harvard Medical School, Boston, Mass.

This is Publication #258 of the Robert W. Lovett Memoriul for the Study of Crippling Diseases.

The studies herein reported have been supported by grants from the Commonwealth Fund, the United States Public Health Service and the Eli Lilly Company.

This lecture was one of a series of 20 Lowell Lectures on “Disease and the Advancement of Basic Science,” delivered at the Massachusetts General Hospital during the months of October, November and December, 1958. This series of lectures was made possible by the imagination of Dr. Henry K . Beecher, Chief of the Anesthesia Service, who conceived the idea, selected the speakers and gained the support of the Lowell Institute.

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SONNECTIVE TISSUE RESEARCH AND THE RHEUMATIC DISEASES 483

I will discuss some of the recent work of our own unit and of other laboratories pertaining to certain connective tissue diseases to illustrate how, by direct and indirect means, clinical observations and concerns may lead to discoveries of fundamental interest.

The group of disorders which has concerned our arthritis unit, primarily, and to which we refer as the connective tissue diseases, are: rheumatic fever, rheumatoid arthritis, disseminated lupus erythematosus, periarteritis nodosa, dermatomyositis and scleroderma. These diseases are systemic in nature, variable in their manifestations and characterized by chronic inflammation of tissues of mesenchymal origin. The most dramatic evidence of connective tissue involvement is seen in various phases of some of these chronic crippling diseases.

The concept of a “connective tissue disease” was introduced by Klinge in relation to rheumatic fever. Prior to this, the fact that rheumatic fever involves such remote organs as the skin, joints and heart was a mystery which was explained neither by the organ pathology of hlorgagni nor by the sub- sequent tradition of Virchow, which held that the cells were the chief partici- pants in disease. Klinge’s observation that the characteristic pathologic changes of this illness are due to alterations in the fibrous connective tissue of the organs affected led him to postulate that fibrous connective tissue comprised a system which could be the site of primary disease. This orientation of our views concerning the pathology of rheumatic fever and periarteritis nodosa and later rheumatoid arthritis provided the pathologist with a plausible explanation of the widespread organ involvement. This concept also found application in the elucidation of other systemic diseases in which no common denominator had been found. Klemperer and his colleagues explained the diffuse nature of disseminated lupus erythematosus in like manner. They, however, stressed the striking alterations in the collagenous portion of the fibrous connective tissue and coined the term, “collagen disease.” They were fully aware that the collagen changes were but one morphologic characteristic of the underlying morbid disease process. Because the term “collagen disease” implies that the changes occur primarily in collagen fibers, it is misleading. We prefer the more inclusive yet, not wholly satisfactory term, “connective tissue diseases,” when referring to this group of disorders.

This ill-defined group of syndromes exhibits in varying degree any or all of the following histologic changes:

1. Inflammation involving blood vessels, the loose or dense connective

2. Edema or staining changes in the intercellular, interfibrillar ground

3. Increased proliferation of connective tissue elements-cells, fibers

4. Fibrosis, resulting in deformities and limitation in function of the

tissue, or all three.

substances or in the fibers themselves.

or ground substance.

musculoskeletal system or of the heart blood vessels.

WALTER BAUER 484

These histologic alterations may be localized to an organ or other structure, or may be generalized. Rheumatoid arthritis, for example, was, until relatively recently, considered to be primarily a disease of joints. Now we realize that lesions in the heart, skeletal muscle, peripheral nerves, skin, eyes and blood vessels are a relatively common occurrence.

As mentioned previously, the causes of these nonspecific inflammatory diseases are quite obscure. The most specific etiologic clue thus far is the very strong evidence that rheumatic fever is related in some way to infection with the beta hemolytic streptococcus. However, the exact relationship be- tween the streptococcus and this disease is entirely unknown. Some workers advocate an immunologic phenomenon. Others entertain the possibility that the so-called pleuropneumonia form of the beta hemolytic streptococcus might be implicated. Much new knowledge of a fundamental nature has resulted from this stimulus, some of which I shall discuss later.

Whether or not other members of this group of diseases could be caused by infection with other organisms, we do not know. One wonders, for example, whether a virus might be involved. So far, inadequate studies have been carried out to exclude this possibility. As with rheumatic fever, many in- vestigators are concerned with the possibility of an immunologic factor. Others point to the possibility of a metabolic defect, or n hormonal one, perhaps related to some type of stress.

In short, no one mode of pathogenesis seems sufficiently probable to merit concerted pursuit to the exclusion of others. All possible etiologic leads should be explored, because of the magnitude and importance of the many prob- lems relating to these chronic crippling diseases.

One fact appears certain, namely, that the connective tissue itself is a prime target of the process and the major site of the manifestations of these diseases. But the nature of its involvement and how it is altered so as to cripple the patient are unknown. It is thus apparent that we are in need of information concerning the nature and behavior of the tissues afflicted.

Most fundamental studies on the nature of connective tissues have been segregated for operational convenience into categories dependent on the tissue components, principally the fibrous proteins, the intercellular, inter- fibrillar ground substance and the indigenous cells.

The major fibrous component of the connective tissue, collagen, accounts for about 30 per cent of the total protein of the mammalian body and is by far the most extensively studied-at first primarily by zoologists and chemists interested in leather, but more recently the properties of this protein have stimulated considerable interest in molecular structure and morphogenesis at the molecular level. The pioneering x-ray &&action studies of Wohlich, Ast- bury, Faure-Fremiet, Bear and others have demonstrated that collagen is widely distributed throughout the animal kingdom. Studies on solutions of collagen by electron microscopy and physicochemical methods have re- vealed the size and shape of the molecule. X-ray diffraction analysis, cor- related with studies on the amino acid composition, have provided the neces- sary information to deduce the intramolecular structure of this material. It is thought to consist of three polypeptide chains, each of them a helix, wrapped

CONNECTIVE TISSUE RESEARCH AND THE RHEUMATIC DISEASES 485

around each other in a superhelix. The molecule itself, at least in the soluble portions of mammalian and fish collagen, appears to be a rigid rod of the order of 3,000 A units in length and about 15 A units wide. Intensive in- vestigations are now underway on the particular sequence of amino acids within the polypeptide chains and the relationship of these sequences to the detailed molecular structure of the protein.

The ultimate results of these investigations are, of course, not completely predictable. However, one can see how such information will be of con- siderable importance in the final unravelling of the structure of proteins, in this case the group of fibrous proteins, and may provide important tools for the ultimate determination of the mechanism of synthesis of these bio- logically important macromolecules.

A particular aspect of the study of collagen which has intrigued Dr. Jerome Gross in our unit, and other investigators, has been the solubilization of collagen and its reconstitution in fibrous form from solution. A factor which has contributed greatly to the recent rapid advances in our understanding of this process was the discovery of the periodic fine structure of collagen with the electron microscope in 1942 by Drs. Hall, Jakus and Schmitt at the Massachusetts Institute of Technology, and independently by Dr. Wolpers in Germany. The electron microscope has revealed in most cases that the fibrils possess a characteristic repeating band pattern of about 640 A units

Early in the century, it had been observed that tendon and skin collagen could be dissolved in dilute organic acids and reprecipitated as fibrils in the test tube simply by neutralization. These solutions, prepared by dissolving a collagenous tissue such as rat tail tendon or calf hide in dilute acetic acid, are clear and viscous, and usually contain little else than dissolved collagen (figs. 2 and 3) . The addition of 1 per cent sodium chloride or neutralization of such solutions results in the immediate formation of a fibrous precipitate exhibiting structure similar to that of the original collagen from which it was derived (fig. 4). These observations were first made by Schmitt, Hall and Jakus in 1942 and marked the beginning of a vigorous and intensive effort to determine the structural characteristics of the molecule of collagen and the forces involved in aggregating these molecules into the specific structure of the native fibrils.

The first hint as to the size and shape of the molecules came through studies of Highberger, Gross and Schmitt on several specifically ordered structural forms precipitated from solutions of collagen. Careful examination of these several different structural types of collagen led these investigators to conclude that the collagen molecule was a rod like particle of the order of 2500 A in length and less than 50 A in width, which they named tropo- collagen. Subsequent physical-chemical studies by Boedtker and Doty at Harvard and by Hall at the Massachusetts Institute of Technology, using the electron microscope, demonstrated that the collagen particles in solution did indeed have essentially the dimensions described by the above investigators. It thus appeared that the collagen molecules in solution, when presented with the proper ionic environment, can spontaneously aggregate in a highly

(fig. 1) .

486 WALTER BAUER

Fig. 1.-In this figure, an electron micrograph of the human skin, the collagen fibrils are arranged in bundles, and the characteristic rcpeating pattern is evident. The bands that one sees are not discs but are the result of the lateral interaction of long, fibrous collagen molecules.

ordered manner and that the type of organization is dependent on the very specific configuration of the molecule itself. Indeed, it is possible by this means to produce collagen precipitates not only as simple fibrils, but also as highly ordered sheets in two dimensions and in some cases in three dimen- sions. One wonders whether such structures, if present in the ground sub- stance, might furnish the proper orientation of the matrix for direction of cell migration during embryogenesis.

This phenomenon of collagen reconstitution may have broad biologic im- plications. One might speculate that this seemingly simple phenomenon, which appears to take place even in the absence of cells or enzymes, may be a model for the formation of complex structures from large molecules both inside and outside of the cells.

Another advance which has facilitated studies on the process of fibro-

CONNECTIVE TISSUE RESEARCH AND THE RHEUiMATIC DlSEASES 487

Fig. 2.-This figure shows a fibril of collagen beginning to dissolve in the dilute acid. Notice the spreading apart of the fibrous elements which comprise the fibril.

genesis and which also provided a tool for the investigation of the metabolism of collagen was the discovery by Gross, Highberger and Schmitt that collagen may be extracted from fresh connective tissue with cold neutral salt solution at physiologic pH and ionic strength. The collagen so extracted can be con- verted to fibrils indistinguishable from those of the native tissue simply by warming the extract to body temperature. Since this phenomenon can be accomplished in a preparation of highly purified collagen, it was suggested that enzymatic action is not required and that here again the aggregation into a highly specific structural unit depends upon the rigid and detailed organiza-

488 WALTER BAUER

Fig. 3.-Electron micrograph, taken by Professor Cecil Hall at the Massachusetts Institute of Technology, of the actual separate molecules of collagen. The magnifica- tion is 100,OOOx.

tion of the collagen molecule. In the laboratories of A. Neuberger in England and D. S. Jackson, it was then demonstrated that the neutral salt-extractable

orated C1*-labeled glycine at an extremely rapid cid-extracted collagen and the insoluble fraction

which showed very little incorporation. These and other data suggested that this readily soluble fraction was most likely a precursor of the insoluble fibrils.

Further studies have continued to yield data of interest to the physician. For example, Dr. Gross has noted that the time of existence of the collagen fibril in itself contributed to the increasing insolubility of the structure. Collagen fibrils formed in vitro by the thermal precipitation technic became more and more insoluble with regard to cold neutral salt solutions as time went on. This model system is then analogous to that which has been shown to exist in nature: collagen of older individuals is known to be less soluble than that of the young. If the extrapolation may be made from the in vitro model system of the living animal, we have here a truly time-dependent phenomenon which might help us to separate certain features of aging which may not be the re- sult of accident and disease.

A recent experimental indication of the significance of supramolecular organization in tissue development has been furnished by Glimcher, Hodge and Schmitt in studies on the process of bone formation in vitro. These investi- gators observed that true apatite crystal structure appeared in association

CONNECTIVE TISSUE RESEARCH AND THE RHEUMATIC DISEASES 489

Fig. 4.-In this figure, on the left, one sees an electron micrograph of collagen molecules in solution; on the right, the result of adding 1 per cent sodium chloride. The precipitated fibrils have all the detailed periodic structure of the native fibrils.

with collagen only when the fibrils had the periodicity of about 640 A, the native structure. Apatite crystals did not form in vitro on collagen aggregates with any of the other artificially produced structures described above. No doubt this model system is extremely oversimplified, and numerous other factors very likely play a role in in vivo physiologic ossification. However, this simple and fundamental principle might underlie the over-all process.

The problem of wound healing, itself the subject of a mountainous volume of literature, is to a large degree one of the behavior and function of con- nective tissue. Its relevance to the organization of tissues in embryogenesis and regeneration and to the crippling sequelae of a variety of human diseases of which rheumatism is an outstanding example is well recognized.

One might expect that the factors effecting wound closure and the phenomenon of wound contraction would be well understood since the ex- perimental system seems so ideal for handling, but this is hardly the case. It has been accepted dogma, for example, that the granulation tissue in the center of the wound is responsible fur pulling together the wound margins in secondary closure. Very recently, Drs. Grillo, Watts and Gross have demon- strated that the dogma does not hold up; the granulation tissues of a skin wound may be completely removed right up to the wound margin without

490 WALTER BAUER

changing the rate of contraction. They further observed that removal of a very narrow picture frame of tissue underlying the wound edge causes im- mediate distraction of the wound and that only upon reforming the contact between wound margin and tissue base is contraction reinitiated.

This simple new observation in an old, well trodden field poses some ques- tions of interest to the embryologist. What are the forces which cause the wound edge to move against the opposing tension of the surrounding tissue? If it is a cellular migration, how do the cells pull the tissue edge with them? Where do these new cellular accumulations come from? What makes them move into the wound? How does this phenomenon bear on the important question of regeneration of removed tissues?

Although the group of chronic conditions collectively and loosely called rheumatism are frequently referred to as the “collagen diseases,” implying important involvement of collagen, another widely distributed component of the connective tissue, the ground substance, has in recent years commanded the attention of pathologist and medical investigator. This amorphous inter- cellular, interfibrillar matrix fills the space not occupied by cells and fibers and lies in the strategic position between cells and blood supply. All nutrients and waste products moving to and from the cells must pass through this ma- terial which may be in the form of a basement membrane or a diffuse jelly. The ground substance varies in composition and organization from tissue to tissue and probably also with time. It is composed of water, proteins, mucopoly- saccharides and a host of low molecular weight substances in varying pro- portions.

The component acid mucopolysaccharides have attracted wide attention. They are typified by hyaluronic acid, the chondroitin sulfates and heparin. These high polymeric carbohydrates are distributed primarily in the extra- cellular, extrafibrillar spaces of the connective tissue, with the exception of heparin, which seems to be localized to the mast cell. Variations in the distri- bution, amount and properties of the mucopolysaccharides have been found during embryogenesis, and with age. They are influenced by a variety of hor- monal actions, perhaps the most characteristic being that of the thyrotropic hormone and steroids. They are altered in a number of pathologic conditions, the most obvious one being rheumatoid arthritis, in which there is a break- down of the hyaluronic acid of the synovial fluid. These substances are always associated with new formation of connective tissue and seem to diminish considerably as the tissue matures and ages.

From the physiologic standpoint, the tissue distribution of mucopoly- saccharides has stimulated considerable interest, since they appear to be localized between the blood vessel and the cells of the parenchymal organs throughout the body. It has been widely speculated that these substances may produce a selective diffusion barrier, at least under certain conditions, to the passage of metabolites and breakdown products between the cells and the blood. Since the distribution and properties of the mucopolysaccharides are known to alter under a variety of conditions, the speculation has been further advanced that such alterations can produce considerable changes in the func- tioning of the parenchymal organs.

CONNECTIVE TISSUE RESEARCH AND THE RHEUMATIC DISEASES 491

Indirect evidence has been obtained to suggest that such interferences in diffusion may actually take place in certain specific tissues. For example, it is observed by Cogan and co-workers that the rate and extent of diffusion of low molecular weight substances through the intact cornea was dependent upon the proportion of polar and nonpolar groups on these substances and that both the epithelium and the stroma were important in the transport of these materials. Paulson et al. in Sweden also observed that diffusion of low molecular weight substances such as N-acetyl glucosamiiie or the neutral sugars were not interfered with by the components of nucleus pulposis, where- as a charged molecule such as nonacetylated glucosamine was completely blocked in its passage.

These are but the hints of a possibly significant physiologic phenomenon. It will be necessary to demonstrate in a direct manner that such varying con- nective tissue barriers to diffusion actually do exist and do have a modifying effect upon the function of other tissues. Exciting speculations such as these which deal with normal and pathologic physiology have played a considerable role in instigating a large amount of investigation into the chemical nature and physical properties of the acid mucopolysaccharides.

In general, these substances may be characterized as linear polymers of N-acetyl hexosamine and a uronic acid or hexose or other sugar. Their as- sociation with protein is usually weak, and they are readily separated from them, in contrast to the glycoproteins such as those found in the serum in which the carbohydrate moiety cannot be readily separated from protein. There are now several known families of mucopolysaccharides. Hyaluronic acid appears to be a linear, high molecular weight chain, consisting of alternat- ing units of N-acetyl glucosamine and glucuronic acid. It appears to differ in some respects, particularly with molecular weight, from tissue to tissue. The chondroitin sulfates are characterized by the presence of N-acetyl galacto- samine plus a uronic acid, in most cases again glucuronic acid. The distribu- tion of these three chondroitin sulfates varies from tissue to tissue and also appears to change with age. A third class of compounds are the heparins, of which there seem to be a variety of species which vary primarily by the number of sulfate groups. These are composed of glucosamine and glucuronic acid, but the amino group of the amino sugar, rather than the N-acetyl group, is very frequently sulfated. These compounds, as is well known, are important in the blood clotting mechanism.

Three major lines of investigation on the acid mucopolysaccharides require further exploration: (1) their distribution in the tissue, their isolation and purification; (2) chemical and structural characterization of the high poly- meric molecules; and ( 3) the synthetic and degradative mechanisms involved in their metabolism. In our own laboratories, Dr. Roger Jeanloz and colleagues are engaged in an intensive study of the chemical composition and sequence of the sugars of hyaluronic acid and chondroitin sulfates ( A and B ) . During the past six years, a systematic attack on the order and linkages of the monomer sugars has been made by the classic methods of methylation of the intact polymer followed by degradation into the individual sugars and intermediate compounds and identification of the methylated products by chromatography,

492 WALTER BAUER

crystallization, et cetera. In order to determine the actual sequence of sugars and the linkages involved, it was necessary to synthesize all the methyl deriva- tives of the individual sugars, namely, hexosamine and glucuronic acid, so that a library of reference compounds might be established. Thus far, all these derivatives have been synthesized, the structure of hyaluronic acid is partly established and considerable progress has been made with two of the chondroitin sulfates. Such studies are essential in order to correlate com- position, structure and function.

With regard to the metabolism of the mucopolysaccharides, pioneering studies of Dorfman and colleagues have made it possible to establish the origin of the 14 unique carbon atoms of the monomer disaccharide of hyaluronic acid. Shortly before, Dr. Luis Leloir discovered and characterized the co- enzymes involved in the synthesis of certain complex carbohydrates, namely, uridine nucleotide conjugates such as uridine diphosphoglucose and uridine diphospho N-acetyl glucosamine. Leloir also showed how glucosamine was converted to galactosamine. Dorfman and colleagues have now identified the uridine conjugate intermediates in hyaluronic acid synthesis in cultures of streptococci. Although much has been learned concerning these low molecular weight intermediates, the actual mechanism of synthesis of hyaluronic acid is still not clear.

Recently, there has been an actively growing interest on the part of the chemist in the congenital abnormalities affecting the connective tissues. Hurler’s syndrome is an excellent example. In this disease, there is an increased accumulation of intracellular mucopolysaccharides in many of the organs of the body, together with an overflow of these substances into the urine. In Hurler’s syndrome at least two acid mucopolysaccharides have been isolated, one of them chondroitin sulfate B; the other is considered to be one of the heparin sulfates. Patients with this disease excrete these two mucopoly- saccharides in large amounts but in varying ratios. The organic chemist now has the tools for characterization of these interesting compounds, and the biochemist is currently studying their synthesis.

The biologic significance of the acid mucopolysaccharides is still a matter of conjecture. Do these reactive high polymers have an ion exchange function in the tissues? What role do they play in calcification and ossification? How do they function in the embryonic organization of connective tissue on a mole- cular and tissue level? Are they involved in fibrogenesis of collagen, in tissue hydration, elasticity and tensile strength? It is known that hormones influence the distribution and actual content of these substances in a variety of tissues. Similar changes are observed in the aging process and in some disease states. Do such alterations influence the over-all physiology of the organism? These questions which deal with fundamental life processes may be closer to solution as a result of the stimulus provided by interest in the rheumatic diseases.

Another large group of amino sugar-containing substances which have recently attracted considerable attention are the glycoprotein and glycolipo- proteins of the blood and tissues. These are proteins to which carbohydrates are covalently bound. Here again, observations in the clinic have lead to an increase in interest on the part of the basic investigator. It is known, for ex-

CONNECTIVE TISSUE RESEARCH AND THE RHEUMATIC DISEASES 493

ample, that there is a large increase in hexosamine-containing proteins in the blood associated with a variety of acute and chronic disabilities, among them the rheumatic diseases. The meaning of these fluctuations is not presently understood.

Thus far, the only glycoproteins with known physiologic function are cer- tain of the hormones, in particular the thyrotropic hormone, some of the blood group substances and also enzymes such as alkaline phosphatase. The physiologic functions of many of the glycoproteins isolated from blood and tissues remain unknown.

One of the very interesting glycoproteins is fetuin, a substance which in the bovine embryo accounts for as much as 50 per cent of the total blood protein. Within several days after birth, this substance diminishes to a very small fraction of the total protein. This remarkable change in quantity still is un- interpretable with respect to physiologic function.

During the past few years, D. Karl Schmid in our unit, using the classic methods of protein fractionation developed in the laboratory of Dr. E. J. Cohn at Harvard, has been actively engaged in the isolation and characteriza- tion of the glycoproteins from blood and tissues. He has isolated the serum alpha 1 acid glycoprotein, now known as orosomucoid, crystallized it and characterized it both physically, chemically and in terms of composition. He has also isolated the complex of alpha-2 glycoproteins and separated them into their component fractions. These have been characterized by physico- chemical and immunochemical technics as well as by sugar and amino acid analysis. A major effort is being made at this time to separate the carbohy- drate from the protein moiety to determine the nature of the linkage that holds the two together. Thus far, these glycoproteins have not yet been iso- lated from connective tissues, although there is a strong suspicion that they are present.

Here again, the clinical observations and intuitive judgments of the clinicians as to significance has caused considerable investigative activity with re- gard to the characteristics and functions of this large group of very recently recognized substances.

More directly related to the rheumatic diseases are the alterations produced in the synovial fluid, involving both protein and mucopol ysaccharide com- ponents, particularly in rheumatoid arthritis. In this disease, there occurs a loss in viscosity of the joint fluid which i s due either to depolymerization of hyaluronic acid or to the production by the synovial cells of low molecular weight mucopolysaccharide. Corticoid therapy produces a dramatic increase in synovial fluid viscosity, often greater than that of normal fluid. Observa- tions such as these have led to studies on the synthesis of hyaluronic acid in tissue cultures of synovial and other connective tissue cells. This isolated cell system has made possible the study of the formation and breakdown of the mucopolysaccharides and the investigation of the mechanisms for hor- monal control of synthetic degradative processes.

The proteins of the ground substance, which in many cases represent a larger proportion of solid than do the mucopolysaccharides, have been practically ignored, with the exception of the synovial fluid proteins. Schmid

494 WALTER BAUER

in our unit, by painstaking fractionation studies, has demonstrated their similarity to the serum proteins. He has also observed significant differences in concentration and distribution of certain proteins between the blood and synovial fluid of normal individuals which are not maintained in the rheumatoid joint. These observations on synovial fluid proteins are providing the experi- ence required for the isolation of ground substance proteins from more complex connective tissues.

Thus, much of the work of our unit has focused around a study of con- nective tissue, its physiology and its chemistry, both in the normal and in various diseases. This work, while “disease oriented,” is properly called basic in that it deals with fundamental chemical and biologic problems with- out pressure for practical results. As more knowledge is obtained, the relation- ships to disease processes may become evident.

A great deal of our laboratory research, however, stems from our daily clinical work. That is, it starts with observations of immediate clinical im- portance, and then proceeds into the laboratory. Let me cite a few examples.

In 1948, when Dr. Rose and his colleagues were testing the serum of a patient convalescing from rickettsial pox, for complement fixation, an unusual observation was made; one serum greatly enhanced the agglutination of sen- sitized sheep erythrocytes at a titer that was 128 times greater than that against unsensitized cells. It was then found that the patient who was initially tested because of rickettsial pox also had rheumatoid arthritis. Dr. Charles Ragan, because of his interest in rheumatic diseases, appreciated the potential importance of this observation in the study of rheumatoid arthritis, and soon the Rose-Ragan Differential Sheep Cell Agglutination Test was introduced. Many modifications of the test appeared during the following years, most of them utilizing erythrocytes.

In 1956, Singer and Plotz introduced the latex fixation test. The chemically inert polystyrene latex particle was used as a carrier and indicator, thus re- placing the erythrocytes previously used in the agglutination tests for rheuma- toid arthritis. This test did not require heat inactivation or absorption of test sera since complement and heterophile antibodies did not interfere in this system.

One year ago, Dr. Schubart, working in Dr. Calkins’ laboratory, suggested that heat-labile components, such as complement, might play some role in the agglutination of particles by the rheumatoid factor. He realized that the latex fixation test provided an ideal tool for such a study, since it could be performed on native serum, rather than inactivated serum. It soon became apparent that this hunch was correct. Sera which had been inactivated prior to running the test yielded stronger and more consistent agglutinations than did parallel aliquots of native serum. Furthermore, many sera which yielded a negative reaction in the native state became strongl:. positive reactors follow- ing heat inactivation. These and other observations. led Dr. Schubart to con- clude that there exists in the serum of patients with rheumatoid arthritis both a factor which facilitates the agglutination of particles coated with gamma globulin and another factor which appears to be a component of complement, which antagonizes the agglutination in some way.

CONNECTIVE TISSUE RESEARCH AND THE RHEUMATIC DISEASES 495

Meanwhile, investigators at other institutions have provided further in- formation concerning this agglutination-activating factor found in many pa- tients with rheumatoid arthritis. These data suggest that the so-called rheuma- toid factor is a gamma globulin of high molecular weight (so-called 19 S globu- lin), which, in vivo appears to combine with a component of the more usual 7 S gamma globulin to yield an enormous component with the sedimentation constant of 22 S. Furthermore, clinical studies of patients with high titers of this factor have shown that these patients have peripheral rheumatoid arthritis rather than spondylitis, and that the highest titers seem to occur in thbse patients who have nodules, especially if accompanied by splenomegaly, anemia, leukopenia and vasculitis.

Here there are many exciting avenues for study. What is the true nature of the complex of 19 S and 7 S globulins which yields the 22 S component? Is it an antigen-antibody reaction, or is it a nonspecific phenomenon? What is the nature of the role of complement in this system? These data raise other questions: Is rheumatoid arthritis a single disease, or is it a group of diseases? Might we, by segregating certain syndromes, be able to come closer to an understanding of the pathogenesis in at least a few cases? Is the rheumatoid factor an incidental accompaniment of rheumatoid arthritis, or might it pos- sibly have some pathogenic significance? If the latter were true, what, if any, physiologic role might it play?

All these questions are at present under active study by members of our unit. Whether or not they result in any data of clinical significance, they are leading to some potentially important basic studies and provide one more il- lustration of how clinical observation may lead to physiologic investigations- and vice versa.

A second example of the way in which clinical problems lead to basic investigation is the study of amyloidosis by Dr. Calkins and his associates. Amyloidosis, an important clinical entity, is found at autopsy in approximately one-third of all patients dying with rheumatoid arthritis. It is also a very common complication of paraplegia, presumably due to the severe decubitus ulcers, and is the chief cause of death in this group of patients. It is a com- mon concomitant of multiple myeloma, and it is seen in some patients with- out known predisposing cause. Despite considerable knowledge concerning the clinical pattern of this disease, very little is known of its chemical nature and nothing of its pathogenesis.

The first problem was to determine something of the chemical and physical nature of amyloid. Dr. Calkins, together with Dr. Robert Giles and later Dr. Cohn, collected amyloid-laden tissues from eight patients with various diseases dying of amyloidosis. Chemical studies have shown that the amyloid is pre- dominantly protein in nature. Amyloid-laden organs have a slightly higher hexosamine and sialic acid concentration than comparable normal tissues. Immunochemical analyses have indicated that this protein is not gamma globulin. On histologic examination, amyloid has a characteristic birefringence snd autofluorescence. Studies using the electron microscope have suggested that it may have an inherent structure, since filamentous threads are detectable within its substances.

496 WALTER BAUER

In a second phase of this work, amyloidosis has been induced in rabbits by means of serial injections of casein. Serial studies of the blood of these rab- bits during the induction of the disease have shown that the presence of a high serum hexosamine concentration, early in the course of the injections, has almost invariably heralded the development of severe amyloidosis. The as- sociation of serum hexosamine-containing components with the development of amyloidosis was supported in a study of 15 patients with various types of amyloidosis by finding a significant elevation of serum hexosamine con- centration in all. In the rabbits, serial electron microscopic studies of the spleen during the induction of the disease have shown that at the time when the serum hexosamine concentration becomes markedly elevated, there is an apparent thickening of the reticulum of the spleen. The nature of these re- lationships is currently under investigation.

Illustrative of an etiologic aspect of the rheumatic disease problem is a continuing bacteriologic study which began in the late 1890’s when Nocard cultured a microbe that had characteristics which distinguished it from any previously described bacterial group. This organism was shown to be the causative agent of a necrotizing pneumonia and pleurisy in cattle, a disease known as bovine pleuropneumonia. The causative agent derives its name from this latter malady. As other organisms possessing similar characteristics have been isolated from other diseases, they have come to be known as pleuropneumonia-like organisms, even though most of them are associated neither with pleurisy nor with pneumonia. They are of particular interest to the clinician engaged in the study of rheumatic diseases because of their un- usual predilection for joint tissues. To date, a variety of isolated strains and species of pleuropneumonia-like organisms associated with arthritis have been isolated from cattle, sheep and goats, rats, mice and fowl. In each instance, it would appear that there are separate strains or species of microbes associated with the different species of animal having the disease. Although some evidence has been adduced that Reiter’s syndrome in humans may be associated with infection by pleuropneumonia-like organisms, it is not sufficiently conclusive to assign a causative role.

There are several distinguishing features which set this group of organisms apart from other established classes of microbes. In general, these organisms are of smaller size than the bacteria, although many strains are highly pleomorphic and produce a mixture of forms which vary in size from small granules of 0.2 to 0.3 P to larger disc-shaped forms that may be several microns in diameter (fig. 5). Filtration experiments have shown that the essential reproductive unit is approximately 0.2 p in its smallest dimension. Thus the pleuropneumonia-like organisms are comparable in size to the larger viruses and rickettsia. However, in contrast to the latter groups, the pleuropneumonia- like organisms grow on lifeless media.

An interesting development occurred in the mid-1930’s when Dr. Louis L. Dienes discovered that under some circumstances, bacteria could undergo an alteration in form and in consequence exhibit most of the important character- istics of the pleuropneumonia-like organisms. These altered forms, known as the L forms of bacteria, have been isolated from a large number of bacterial

CONNECTIVE TISSUE RESEARCH AND THE RHEUMATIC DISEASES 497

Fig. 5.-This figure shows very early growth of a beta streptococcus L colony, with small granules and large bodies. x about 2,600.

species. It is particularly noteworthy that in this altered state, the L forms of bacteria are capable of maintaining growth and reproduction. It is further noted that under appropriate conditions, the normal bacterial form has been regained from L forms of many bacterial species, even after a large number of subcultures in the L form. It is an interesting consequence of this alteration in morphology that the L form of a bacterium is highly resistant to penicillin, yet when the bacterium is recovered after withdrawal of the antibiotic, the bacterial sensitivity, in most instances, is not much altered.

For this reason, a study of the L forms and pleuropneumonia-like organisms has been conducted by Dr. John T. Sharp in an attempt to elucidate the mechanism of action of penicillin. To date, he has discovered that the L form of the beta hemolytic streptococcus lacks the cell wall polysaccharide of the parent organism. This finding suggests that the absence of this cell wall con- stituent accounts for the pliability and fragility of the L form of bacteria. Further, it would seem reasonable to speculate that this structural deficiency is a direct result of exposure to penicillin, the procedure used in obtaining the L forms.

498 WALTER BAUER

Although no role in human disease has been discovered for L forms of bacteria, several interesting possibilities are being explored. Because of the similarity of the L forms to the pleuropneumonia-like organisms and the pre- dilection of this group of organisms for articulations, Dr. Sharp is investigating the possible role of the L form of the group A hemolytic streptococcus in rheumatic fever. As stated previously, there is abundant evidence that the group A hemolytic streptococcus is related in some specific manner to the initiating of attacks of rheumatic fever. However, most attempts at isolating the bacterium from the diseased tissues have been unsuccessful. Furthermore, the mechanism whereby the streptococcus precipitates the disease remains a mystery. With this in mind, it has been of particular interest that the group A hemolytic streptococcus is capable of growing and reproducing in an L form. Attempts both in our laboratories and those of others have been un- successful in isolating L forms of the streptococcus from either body fluids or tissues of rheumatic fever patients. To date, we have no way of knowing whether these failures indicate the absence of this organism in the disease or whether the conditions for cultivation have been inappropriate. Although we have no evidence that the L form of the streptococcus plays any role in rheumatic disease, it is of interest that the presence of a living agent in the diseased tissues in rheumatic fever is compatible with all the known facts. The persistence of a living agent in this disease could readily explain the heightened immune response to the streptococcus, the persistence of micro- scopic lesions for years after other evidence of activity has subsided and the recently observed postvalvulotomy syndrome. The persistence of an altered bacterial form is also compatible with the current concept that immune mechanisms play an important role in the pathogenesis of the disease.

If, how and when the reported studies pertaining to the nature of connective tissue will lead back to the rheumatic diseases is not even a matter for con- jecture at this time. Nor can we predict where the pursuit of problems of clinical significance will lead us, or when information will be forthcoming which will aid us in the alleviation of the rheumatic diseases. However, the “disease orientation” has done much to stimulate and support an area of fundamentaI research which has enriched our store of biologic knowledge. It is also our belief that studies of the type reported, carried on freely in a medical environment, will be a source of continual stimdation of ideas and activities relating directly and indirectly to human biology and disease.

Walter Bauer, M . D., Jackson Professor of Clinical Medicine, Harvard Medical School, Boston, Mass.; Chief of the Medical

Services, Massachusetts General Hospital.