Philip D. McMaster

CONDITIONS IN THE SKIN INFLUENCING INTERSTITIAL FLUID MOVEMENT, LYMPH FORMATION, AND LYMPH FLOW

Part of the fluid which escapes from the blood vessels into the tissues returns to the blood directly, part enters the lymphatic capillaries to become lymph. Little is known about the formation and flow of lymph, that is to say, of the factors influencing the movements of interstitial fluid through the tissues into the lymphatics and of the conditions favoring or hindering the flow of fluid in the vessels themselves. To throw some light on these subjects, studies were made of the movements of fluid introduced into cutaneous connective tissue, and of the pressure relationships within the lymphatic capillaries and in the tissues outside these vessels. Techniques have also been devised to determine changes in lymph flow in living human and animal skin and to observe some of the activities of the lymphatics, in the repair of injuries and in the defense of the body against infection. For the present paper, we have selected from these studies the findings which are most closely related to the problems under discussion in this conference. Much of the work has already been published; the remainder stands unfinished and incomplete, owing to sudden interruption, more than five years ago, by the approach of the recent world catastrophe.

The Source of Fibrinogen.

The formation of fibrinogen has been ascribed to many tissues and organs of the body. Whipple, 1 Goodpasture 2 and others suggested that this substance takes its origin from the liver and intestinal tract; and recently Foster and Whipple 3 have presented excellent evidence that the liver is the main if not the sole source of fibrinogen. Schultz, Nicholes and Schaefer 4 have produced corroboratory data. Final proof in the matter has waited, however, upon the findings in liverless animals. In 4 hepatectomized rabbits 5 we have determined the concentration of blood fibrinogen before operation and at various periods thereafter. A postoperative decrease in the substance was always found. In 6 others a partial depletion of fibrinogen was accomplished after hepatectomy and its concentration in the blood was followed until death. Controls subjected only to defibrination were also studied. The initial defibrination was accomplished under ether anesthesia by slowly injecting into the jugular vein 100-150 cc. of compatible defibrinated blood while removing an identical quantity from the carotid artery. The procedure was repeated 4 or 5 times. This reduced the blood fibrin concentration to one third to one fifth its original amount. By continuing the blood exchange a still greater reduction was sometimes brought about in the controls. Fibrinogen was estimated in the form of fibrin by the method of Foster and Whipple 6 as modified by Schultz, Nicholes and Schaefer. 4 In the controls fibrinogen regeneration was exceedingly rapid. Within 5 or 6 hours after a 90% reduction, a complete return to the previous amount was observed, regeneration far more rapid than has been reported for the dog. 7 Twenty-four hours later the concentration had reached a level 50% above the normal figure and in two instances it had doubled within 48 hours. The findings were wholly different in the liverless rabbit. In 6 animals blood fibrin determinations were made immediately after the hepatectomy and then the partial defibrination was carried out, reducing the circulating amount by 65 to 80%.

Formation of Agglutinins Within Lymph Nodes

Experiments were planned to determine whether or not lymph nodes form antibodies against antigens injected into the peripheral lymphatic capillaries. On 2 successive days killed cultures of B. paratyphosus-B were intradermally injected into the right ear of a large series of mice. The cervical lymph nodes on the right side became much enlarged while those on the left side appeared normal. At varying intervals up to 12 days after the last injection groups of 10 animals were etherized, bled for serum and the lymph nodes which drain the lymphatics of the ears excised. The right nodes were pooled in one group, the left in another. They were ground with sand, extracted with Ringers solution and the extracts and serum titrated for agglutinins by 3 methods, the microscopic, the macroscopic and the centrifugation-macroscopic method of Gates. 1 On the second, third and fifth days after the last injection no agglutinins could be demonstrated in the node extracts or sera; but on the seventh day agglutinins were strongly positive in the extract from the nodes on the injected (right) side at a dilution of 1 in 120. They were questionably positive in the serum at a dilution of 1 in 30. The agglutinin titre was increased in both materials as procured on succeeding days, remaining slightly stronger in the node extract. At length, on the twelfth day, antibodies appeared in the extract from the nodes on the uninjected side.

The Fixation of Certain Viruses on the Cells of Susceptible Animals and Protection Afforded by Such Cells

Rabbit embryo tissue was grown in a thin plasma clot and after some days the cultures were submitted to a trypsin solution strong enough to digest the clot and free the cells that had extended into it. 1 By repeated pipettings and filtrations through lens paper, alternated with differential washings in gelatin-Tyrode solution, suspensions were obtained of the living cells as individuals. They were mixed at room temperature with suspensions of virus, and after an interval the cells were recovered with the centrifuge, again washed repeatedly, treated in various ways and inoculated into rabbits. The viruses used were vaccinia and the filterable agent causing the rabbit fibroma described by Shope. 2 Cells exposed to virus and repeatedly washed invariably gave rise to lesions in susceptible animals. The briefest exposure at room temperature permitted by the conditions resulted in an association of the virus with the cells, which withstood many washings of the latter. The fixation thus indicated took place not only upon living cells but upon those killed by heat, ultraviolet light, and water respectively, and often was as considerable. Incubation of the material with immune serum in vitro, followed by repeated washings prior to infection, resulted in neutralization of the virus associated with dead cells, whereas these procedures were without effect when the cells were alive. The possibility that serum antibodies accumulated on the dead cells and were carried into the final inoculum was ruled out by appropriate tests. Suspensions of individual, washed cells procured from cultures of Shope tumor tissue, or from rabbit embryo cultures inoculated with vaccinia, were found to carry the virus.

Agglutinin Formation Within the Lymph Nodes of Resistant and Susceptible Mice

An accompanying paper has reported the formation of agglutinins within lymph nodes following intradermal injection of killed cultures of bacteria in the ears of mice. Earlier work from this laboratory has shown such intradermal injections to be largely intralymphatic. After injections of killed bacteria into the ear of an animal a cervical lymph node draining the lymphatic capillaries enlarges and a week later agglutinins in high concentration can be found within it. At this time the blood shows a lower concentration of agglutinin and the lymph nodes elsewhere in the body yield no demonstrable antibody. It is evident that the procedure engenders, in the early days of the immunizing process, a high concentration of agglutinin in a localized region. We have experimented to find whether the lymph nodes of strains of generally susceptible and resistant mice developed by Webster 1 , 2 , 3 , 4 differ in the ability to form agglutinins to certain bacteria to which they are susceptible and resistant respectively, and to other organisms against which their relative resistance is unknown. Approximately 200 mice of each of the strains mentioned were obtained through the courtesy of Doctor Webster. A similar number of stock white mice of the ordinary laboratory mixed strain were employed for comparison. In each experiment groups of 12–20 mice from each strain were injected in the skin of both ears with killed cultures of various agglutinin forming bacteria, B. enteritides, B. paratyphosus-B or B. prodigiosus, using but one antigen in each experiment. The cervical lymph nodes draining the ears were removed after varying intervals up to 10 days following the last injection, and the animals were bled for serum. The individual nodes from the mice of each group were pooled and extracted and the titre of the extract was compared with that of the serum, and with that of the node extracts and sera of the other groups.

Urobilin Physiology and Pathology

Further evidence is presented, in addition to that of our previous papers, that the intestinal tract is, under ordinary circumstances, the sole place of origin of urobilin. So long as the biliary tract remains sterile the presence of the pigment in bile and urine is entirely dependent upon the passage of bile to the intestine. Animals rendered urobilin-free by the collection of all the bile from the intubated, uninfected common duct, remain urobilin-free during and after extensive blood destruction caused by intravenous injections of distilled water, as also after reinjections of the animals own blood, hemolyzed in vitro. No urobilin appears in the bile, urine, or feces of animals so intubated when blood destruction has been caused by sodium oleate, or by an agent, toluylenediamine, which damages the liver as well as the blood. On the other hand, when bile flow into the intestine is uninterrupted, urobilinuria occurs during blood destruction caused in any of the ways mentioned and it parallels, both in severity and duration, the destructive process. Merely increasing the amount of bilirubin within the intestines of healthy dogs by feeding urobilin-free bile, will lead to marked urobilinuria. The extravasation of blood into the tissues, resulting from the trauma of an operation for intubation of a bile duct, does not lead to urobilinuria in animals losing all of the bile after this operation, but may do so when only a small fraction of the bile is drained, while the remainder reaches the intestine as usual. The production of artificial hematomas, without operation, is not followed by urobilinuria, under the circumstances last mentioned, but merely by an increase in the bilirubin of the bile. The effect on the liver of the anesthetic employed during the intubation may be responsible for the difference in the two cases. During the course of certain intercurrent infections affecting some of the intubated animals, notably distemper, there was a drop in the hemoglobin percentage of the circulating blood, accompanied by an increased output of bile pigment or further by urobilinuria, when the conditions were such that bile still reached the intestine. The findings pointed to increased blood destruction as a factor in the urobilinuria. The evidence presented, taken with that of our previous papers, suffices to demonstrate, that urobilinuria, occurring during blood destruction, is primarily the result of an increased excretion of bilirubin from which, in turn, an unusually large quantity of urobilin is formed within the intestine. The liver fails to remove from the portal blood all of the latter pigment which is resorbed and consequently some of it reaches the kidneys and urine. Our work has been carried out on animals with uninfected biliary tracts and livers, save for one case which has special mention. The influence of infection of the biliary tract on the place of formation of urobilin and the development of urobilinuria will be discussed in a succeeding communication.

The concentrating activity of the gall bladder

In a previous paper we have noted the fact that the fluid which collects in bile ducts experimentally obstructed is an inspissated, tarry bile when the ducts communicate with the gallbladder, whereas in ducts unconnected with this viscus the fluid is thin and soon becomes free from pigment and cholates. It has long been recognized that the gall-bladder must have a concentrating function, since bladder bile is more concentrated than duct bile from the same animal; and continued functioning during stasis will explain the tarry bile then found. The inspissation occurs so rapidly as to raise the question whether concentration of the bile in periods of intermittent or partial stasis may not be an important favoring element in the formation of gall-stones. To determine the rate of concentration advantage has been taken of the arrangement of the hepatic ducts in the dog. There are three of these, which unite to form a common duct, with the cystic duct emptying high up into the central one. Through an opening near the lower end of this last a catheter was pushed into the neck of the gall-bladder, which was emptied and washed with salt solution; and the duct was ligated after the catheter had been withdrawn. The bile from the middle lobes of the liver had now no way of escape save into the gall-bladder. That from the lobes to either side still reached the common duct, but from this it was collected into a rubber balloon placed in the peritoneal cavity. The laparatomy incision was completely closed. The dogs tolerated the operation well. Control experiments in which a second balloon was substituted for the gall-bladder showed that the separated portions of bile differed little in their pigment content, which was taken as the index to concentration.

Effect of the Pulse on Lymph Formation and Interstitial Movement of Substances

The results of recent investigations suggest that connective tissue fibers serve as pathways for the extravascular transport of vital dyes escaping from lymphatics. 1 In unpublished studies on the ear of the mouse, gentle, alternating, external pressures (0 to 2 cm. or 8 cm. of water) caused squeezing of the tissues and bending of these fibers and at the same time the rapidity of spread of vital dyes through the interstitial tissues was increased. That gentle massage increases the lymph flow from a part is well known. 2 The work to be reported now deals with the effect of pulsation of the blood vessels both upon lymph flow and upon the movement of substances through the interstitial tissues. Warmed, defibrinated, aerated rabbits blood was perfused for periods of 1 to 1 1/2 hours, through freshly cannulated rabbits ears, with the use of an apparatus providing pulsatile and non-pulsatile pressures. “Systolic” and “diastolic” pressures of 141/60 mm. of mercury were used in the case of the pulsatile pressure at about 100 “beats” per minute, while for the constant pressure the equivalent of 141 mm. of mercury was employed. It will be seen that a somewhat higher mean pressure was used in the latter instances. Lymph formation and flow were estimated by filling a few peripheral lymphatics near the tip of the ear with a vital dye and noting the movement of the dye colored lymph toward the base of the ear. The method has been previously used in human skin. 2 To study the rate of spread of substances through the tissues, minute amounts (0.01 to 0.02 cu. mm.) of a vital dye (pontamine sky blue) were introduced through micropipettes into the subepidermal connective tissue of the ear by capillarity without utilization of pressure, Camera lucida drawings of the outline of the dye spots were made immediately after placing the dye in the tissues and again half an hour and 1 hour later.

The Lymphatic System

Acknowledgements Series Foreword Introduction The Chemicals and Cells of the Lymphatic System The Lymph System: An Examination of the Lymph Nodes and Lymphatic Circulation in the Body Secondary Lymphatic Organs: An Examination of the Lymphatic Functions of the Spleen, Appendix, Peyers Patches, Tonsils, and Adenoids The Immune Response: Cell Mediated and Antibody Mediated Responses The Development of the Lymphatic System History of Discovery from the Ancients to the 19th Century The Lymphatic System in the 20th and 21st Centuries Diseases of the Lymphatic System: Cancer Diseases of the Lymphatic System: Primary and Secondary Lymphatic Organs Allergies and Autoimmune Responses The Influence of Exercise and Nutrition on the Immune Response Interesting Facts on the Immune System Websites of Interest List of Acronyms and Abbreviations Bibliography Glossary Index

Development of Antiviral Properties within Lymph Nodes.

Every intradermal injection is intralymphatic and infectious agents introduced by way of a skin wound may reach the lymphatic glands directly. 1 , 2 Specific bacterial agglutinins are formed in the nodes draining the ears of mice into which killed cultures of bacteria have been injected intradermally. 3 Experiments now show that the lymph nodes elaborate a principle capable of neutralizing a virus draining to them. Rabbits were inoculated intradermally in one ear with a standard amount of vaccine virus and in the other with typhoid bacterin. Thirty-two experiments were done. In half of these the ears were amputated after an hour, while in the others the ears were left intact. After later intervals of from 2 hours to 15 days the cervical lymph nodes of both sides, inguinal nodes, bone marrow and spleen were removed and extracts of them were inoculated separately into 3 or 6 normal rabbits (as was serum procured from the same animals at the same time) to test for their content of virus (titration tests). In addition neutralization tests were set up to learn whether these extracts possessed antiviral properties. Only the extracts of the ears and cervical lymph nodes of the virus-injected side gave rise to virus lesions. A few hours after the injection of standard amounts of virus in the ears equal amounts of virus were recovered from the lymph nodes. By the second or third day an increase in virus was demonstrable in the extracts of the nodes on the virus-injected side. A rapid decline took place thereafter and by the 7th to the 9th days the extracts failed to produce lesions. At no time did the serum or extracts from other nodes or organs give evidence of virus.