Delayed hypersensitivity is one of several immune responses initiated by thymus-derived (T) lymphocytes. The mononuclear infiltrate of delayed hypersensitivity is a collaborative phenomenon between T lymphocytes and monocytes. This collaboration can be separated into components which I have chosen to call the  three R\u27s  of delayed hypersensitivity. In this discussion I will define these  three R\u27s  and examine the usefulness of this concept in clinical medicine

Horwitz, David

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The "Three R's" of Delayed Hypersensitivity* DAVID HORWITZ, M.D. Associate Professor, Rheumatology Division, Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville Delayed hypersensitivity is one of several im-mune responses initiated by thymus-derived (T) lyrriphocytes. Other functions of T cells are listed in Table . 1 and summarized in reference 1. The mononuclear infiltrate of delayed hypersensitivity is a collaborative phenomenon between T lymphocytes and monocytes. This collaboration can be separated into components which I have chosen t6 call the "three R's'~ of delayed hypersensitivity. In this dis-cussion I will define these "three R's" and examine the usefulness of this concept in clinical medicine. Hematoxylin and eosin sections of skin biopsied 48 hours after intradermal injections of antigen characteristically show mononuclear cells around blood vessels and in connective tissue. As early as 1951, however, Gell and Hinde (2) reported that the monocyte was the principal participant in the skin site. In 1963, McCluskey (3), using radio-isotopic techniques, found that 90% of the mono-nuclear cells in the reaction site were blood-borne and originated from cells that had divided 48 hours previously. Subsequently, cell transfer studies have provided conclusive evidence that the rapidly divid-ing cells originated in the bone marrow and were precursors of the blood monocyte. Tiie evidence is as follows: 1) Injection of immune lymphocytes into nonimmune, irradiated recipients failed to transfer positive skin reactions ( 4); 2) Injection of bone marrow cells from nonimmune donors to sensitized, irradiated animals resulted in positive responses ( 5); 3) Local injection of nonimmune macrophages into the skin of irradiated, sensitized recipients elicited positive responses in these areas ( 6). These studies indicated that two populations of cells are required to elicit delayed hypersensitivity. * Presented by Dr. Horwitz at the 45th Annual McGuire Lecture Series, November 8, 1973, at the Medical College of Virginia, Richmond. MCV QUARTERLY 10(2) : 61 ·64. 1974 The first are radioresistant, which were later defined as long-lived, T lymphocytes. The second population are cells of the monocyte/ macrophage system and are derived from radiosensitive precursors in the bone marrow. From this information we can develop a con-cept of delayed hypersensitivity consisting of three components (Table 2). First, sensitized T lympho~ cytes, perhaps with some help from macrophages, recognize intradermally injected antigen. Secondly, antigenic contact triggers a response in sensitized lymphocytes. Within a few hours these cells release soluble mediators (7) called Iymphokines into the environment and later, activated small lyrriphocytes transform into blasts and proliferate. Thirdly, through the amplifying effects of lymphokines, such as migra-tion inhibitory factor (MIF) and monocyte chemo-tactic factor (MCF), other leukocytes, predominantly monocytes, accumulate at the skin .test site and comprise the reaction of delayed hypersensitivity. This mononuclear infiltrate is similar to that of any other chronic inflammatory reaction. The triggering event, however, is an immunologically specific re-sponse of T lymphocytes. Techniques are available to evaluate each of the "three R's." The number of circulating T cells avail-able for antigen recognition can be quantitated by several methods. The most widely used method at this time is a rosette technique. When sheep erythro-cytes are incubated with human lymphocytes at 4 °C, the erythrocytes form rosettes around T cells ( 8). Secondly, heterologous anti-T lymphocyte serum has been developed (9) and may be used to quantitate T cells either by immunofluorescence or cytotoxicity. The second component, response to antigen, can be evaluated by measuring the lymphocyte pro~ Iiferative response to antigen. In these studies lymphocytes are cultured with test antigen for five-61 62 HORWITZ: "THREE R's" OF DELA YEO HYPERSENSITIVITY TABLE 1 THYMUS-DERIVED LYMPHOCYTE FUNCTIONS 1. Delayed hypersensitivity 2. Activation of macrophages to resist infection 3. Rejection of allografts and tumors 4. Regulation of antibody production by B cells to-seven days and proliferation is assayed by measur-ing incorporation of a radiolabeled DNA precursor. Alternatively, one can measure lymphokine production from antigenically stimulated lympho-cytes. Migration inhibitory factor has received the most attention (7). Various techniques to measure MIF have been developed which are based on the inhibition of leukocyte migration by the culture supernatants of stimulated lymphocytes. The method of Rocklin and David (10) correlates well with delayed hypersensitivity. These workers use guinea pig peritoneal macrophages as effector cells. Other workers have substituted human "buffy coat" leuko-cytes for guinea pig macrophages ( 11 ) . Results obtained with these cells as the indicator cells, how-ever, did not always correlate with cutaneous skin reactivity to antigen (12). I prefer to measure MCF rather than MIF, because both response and reaction can be evaluated. With this technique we can detect mediator produc-tion from a given patient's lymphocytes and also evaluate the capacity of that subject's monocytes to respond to bis own mediator. With a method to measure leukocyte chemotaxis, we can evaluate both lymphocyte and monocyte function. To perform this technique a chamber is divided into an upper and lower compartment by Nuclepore® membrane. Monocytes are placed in the upper cham-ber and the chemotactic stimulants in the lower chamber. The specific migration of cells from the upper to the lower surface of the membrane filter is quantitated ( 13). These in vitro methods can be used to define the component that is defective in patients with impaired delayed hypersensitivity. Lymphocytes from children with a congenitally absent thymus gland (the DiGeorge syndrome and Nezelof syndrome) fail to form rosettes with sheep erythrocytes or to proliferate in response to mitogens. These children, then, do not have sufficient T lymphocytes capable of antigenic recognition. Impaired cell-mediated immunity in chronic mucocutaneous candidiasis may be due to several defects, either .in a response or reaction. Patients with this disease usually have adequate numbers of circulating lymphocytes but may have selective de-fects in lymphocyte or monocyte function. In some subjects, blast transformation is intact but MIF is absent ( 14). In others, mediator production is intact, but these patients' lymphocytes fail to proliferate in response to antigen (15). Finally, Snyderman (16) reported a patient with chronic mucocutaneous candi-diasis whose monocytes responded poorly to a chemotactic stimulus. Although it is tempting to speculate that the defects detected with in vitro TABLE2 DELAYED HYPERSENSITIVITY A COLLABORATIVE PARTICIPATION OF LYMPHOCYTES AND MACROPHAGES IN AN IMMUNOLOGICALLY SPECIFIC INFLAMMATORY REACTION Component Assay System 1. The recognition by small (thymus-derived) lymphocytes of material which is antigenic to the host. 2. A response of sensitized lymphocytes as detected by the release of soluble factors (lymphokines) and later by cell division. Quantitation of T lymphocytes by: 1. Spontaneous rosettes without sheep erythrocytes. 2. lmmunofluorescence or cytotoxicity using heterologous antihuman thymocyte serum. Generation of lymphokines 1. Migration inhibitory factor (MIF) 2. Macrophage chemotactic factor (MCF) Blast transformation and proliferation 3. A mononuclear inflammatory reaction, consisting Monocyte response to MCF predominantly of "activated" macrophages, which "Skin-window" assay leads to the attack, destruction, and sequestration of autologous or foreign materials. HORWITZ: "THREE R's" OF DELAYED HYPERSENSITIVITY 63 techniques accurately reflect an in vivo disturbance, the data must be interpreted with appropriate caution at this time. Delayed hypersensitivity may be impaired in conditions without a demonstrable defect in cellular function . Serum factors inhibit lymphocyte function in certain diseases and several examples are listed in Table 3. The role of these serum inhibitors has not been defined. Moreover, primary disorders of lymphocyte function have not been differentiated from secondary effects caused by serum factor in many diseases with impaired cellular immunity. Recent studies from this laboratory suggest that anergy in such diverse diseases as systemic lupus erythematosus (SLE) and primary intracranial tu-mors ( 17) is caused by serum inhibitors rather than defective cells. Fractionation of inhibitory serum in patients with each of these diseases revealed an IgG factor that broadly suppresses reactivity of allogeneic as well as autologous lymphocytes. This inhibitory lgG factor has been named lymphocyte regulatory immunoglobulin (LRG) and increased LRG in SLE may, in part, explain decreased delayed hypersensitivity. Patients with SLE and other dis-eases also have a lymphocytotoxic antibody that may depress cell function. In summary, I have attempted to develop a working model of delayed hypersensitivity that may be useful in understanding areas puzzling to the non-immunologist. The reader is urged to ask the follow-ing questions in order to evaluate scientific papers dealing with defective cellular immunity in various diseases. First, did the patients studied have adequate numbers of circulating lymphocytes and monocytes? Secondly, did the methods used evaluate a single component of delayed hypersensitivity or were sev-eral components analyzed? Finally, did the authors distinguish a primary cellular defect from secondary TABLE 3 DISEASES WITH SERUM INHIBITORS OF LYMPHOCYTE FUNCTION 1. Hematologic and solid tumors 2. Systemic lupus erythematosus 3. Tuberculosis 4. Multiple sclerosis 5. Hepatitis 6. Lepromatous leprosy 7. Ataxia telangiectasia 8. Multiparous women 9. Multiple transfusions humoral effects? Separation of delayed hypersensi-tivity into "three R's" may be helpful in organizing and understanding present information. REFERENCES 1. WORLD HEALTH ORGANIZATION SCIENTIFIC REPORT: Cell-mediated immunity and resistance to infection. WHO Tech Report No 519:6, 1973. 2. GELL PGH, HINDE IT: The histology of the tuber~ulin reaction and its modification by cortisone. Br J Exp Path 32:516, 1951. 3. McCLUSKEY RT, BENACERRAF B, McCLUSKEY JW : Studies on the specificity of cellular infiltrate in delayed hypersensitivity reactions. J lmmunol 90 :466, 1963. 4. COE JE, FELDMAN JD, LEE S : Immunologic competence of thoracic duct cells. I. Delayed hypersensitivity. J Exp Med 123 :267, 1966. 5. LUBAROFF DM, WAKSMAN BH : Bone marrow as source of cells in reactions of cellular hypersensitivity. I. Pas-sive transfer of tuberculin sensitivity in syngeneic systems. J Exp Med 128:1425, 1968. 6. VOLKMAN A, COLLINS RM: The restorative effect of peritoneal macrophages on delayed hypersensitivity fol-lowing ionizing radiation. Cell Jmmunol 2:552, 1971. 7. DAVID JR: Lymphocyte mediators and cellular hyper-sensitivity. N Engl J Med 288: 143, 1973. 8. JONDAL J, HOLM G, W!GZELL H : Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J Exp Med 136:207, 1972. 9. WILLIAMS RC, DEBOARD JR, MELLBYE OJ, MESSNER RP, LINDSTROM FD : Studies of T-lymphocytes and B-lymphocytes in patients with connective tissue diseases. J Clin Invest 52:283, 1973. 10. ROCKLIN RE, MEYERS OL, DAVID JR: An in vitro assay for cellular hypersensitivity in man. J lmmunol 104:95, 1970. 11. SjijBORG M, BENDIXEN G: Human lymphocyte migration as a parameter of hypersensitivity. Acta Med Scan. 181:247, 1967. 12. KALTREIDER HY, SOGHOR D, TAYLOR JB, DECKER JL: Capillary tube migration for detection of human delayed hypersensitivity : Difficulties encountered with "buffy coat" cells and tuberculin antigen. J lmmunol 103: 179, 1969. 64 HORWITZ: "THREE R's" OF DELAYED HYPERSENSITIVITY 13. HORWITZ DA, GARREIT MA: Use of leukocyte chemo-taxis in vitro to assay mediators generated by immune reactions. J Immunol 106:649, 1971. 14. CHILGREN RA, MEUWISSEN HJ, QUIE PG, Gooo RA, HONG R: The cellular immune defect in chronic mucocutaneous candidiasis. Lancet l: 1286, 1969. 15. GOLDBERG LS, BLUESTONE R, BARNEIT EV: Studies on lymphocyte and monocyte function in chronic mucocu-taneous candidiasis. Clin Exp lmmunol 8:37, 1971. 16. SNYDERMAN R, ALTMAN LC, FRANKEL A, BLAESE RM: Defective mononuclear leukocyte chemotaxis: A pre-viously unrecognized immune dysfunction. Ann Intern Med 78:509, 1973. 17. BROOKS WH, NETSKY MG, NoRMANSELL DE, HORWITZ DA: Depressed cell-mediated immunity in patients with primary intracranial tumors. J Exp Med 136:1631, 1972. 

The  Three R\u27s  of Delayed Hypersensitivity

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