METHODS: A Companion to Methods in Enzymology 9, 305–310 (1996)

Article No. 0035

What Models of Granulomatous InflammationProvide the Immunologist1

Ahmed Metwali, David Elliott, Arthur Blum, and Joel V. WeinstockDivision of Gastroenterology–Hepatology, Department of Internal Medicine, University of Iowa,Iowa City, Iowa 52242

essary to appreciate the function and complexity ofGranulomas usually serve to protect the host from the spread these lesions.

of persistent microorganisms or other enduring injurious sub- Usually, granulomas are protective mechanisms thatstances. They are complex inflammatory reactions that use check the spread of persistent microorganisms (1).many immune mechanisms to control the inciting nudis. These Also, they encase other enduring obnoxious substanceslesions can persist for weeks, months, or even years. Thus, injurious to host tissue (2, 3). Composed of macro-understanding the mechanisms that enhance, diminish, or mod- phages and other inflammatory cell types, granulomasulate granulomas could aid in the treatment of many diseases. form over several days and can persist for weeks,Moreover, experimental animal models of granulomatous inflam- months, or even years. These focal, chronic inflamma-mation allow sophisticated investigation of some disease pro-

tory lesions use various immune defense mechanisms,cesses not possible using human subjects. Granulomatous in-governed by many immunoregulatory circuits, to de-flammations are chronic, composed of activated leukocytes thatstroy or restrain the invading nidus. It is probable thatare selected for deposition at the site of injury. They use variousgranulomas that form in response to each of the manyeffector mechanisms, delicately balanced by many immunoregu-granulomagenic factors employ somewhat differentlatory circuits. Under some experimental conditions, granulomasfactors and immunoregulatory mechanisms to limit in-can be isolated readily from host tissue and subjected to sophis-jury. Yet granulomas themselves cause tissue damage.ticated immunological analysis. Therefore, it is possible to dis-Exuberant granulomatous responses may induce ex-sect the actual, local control mechanisms that govern the granu-

lomatous response. Lesions learned studying granulomas are tensive fibrosis and permanent organ damage.applicable to other types of inflammation, since granulomas use Granulomas are classified into two broad categoriesimmunoregulatory networks and soluble cytokines common to (Table 1). The first group comprises the foreign body-many inflammatory states. Using these experimental models, it type lesions that form in response to persistent irri-is readily apparent that studies utilizing splenocytes or periph- tants, which induce inflammation without immunologi-eral blood leukocytes may not reveal the true, dominant immuno- cal memory. These granulomas are structurally simple,regulatory mechanisms employed at sites of active inflamma- and the cellular elements usually turn over slowly.tion. The leukocytes of spleens and blood are a mixture of cells The second group consists of the hypersensitivityin various stages of activation, many of which are not destined granulomas, caused by persistent antigens that induceto participate in a selective immune response. Also, granulomas anamnesis and delayed hypersensitivity reactions.are sustained inflammatory reactions permitting analysis of the

Most granulomatous diseases of major pathological sig-unique features of chronic maintenance, as opposed to acutenificance form hypersensitivity-type granulomas. Thisphase inflammation. q 1996 Academic Press, Inc.group of lesions is more dynamic and cellularly complexthan foreign body granulomas. They can display rapidstructural remodeling and cellular turnover.

The profile of cytokines made within hypersensitivitygranulomas allows their further subclassification. Hy-

Granulomas are a consequence of the normal im- persensitivity granulomas that are effective at control-mune response. To perceive how granuloma models can ling invasive microorganisms frequently produce largehelp us understand the immune system, it is first nec- amounts of IFN-g, TNFa, and other Th1-like cytokines

(4–7). These cytokines heighten macrophage activa-tion, promoting the death of pathogens harbored intra-1 Grants from the National Institutes of Health (AM38327 andcellularly (8, 9). Alternatively, lesions responding toDK07663) and the Crohn’s and Colitis Foundation of America, Inc.,

supported this research. parasite ova and other less invasive factors may pro-

3051046-2023/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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306 METWALI ET AL.

duce predominantly IL4 and IL5 (10). These cytokines hypersensitivity responses are associated with granu-loma formation. Also, there are granulomatous dis-favor eosinophilia and B-cell development typical of al-

lergic-type responses. ‘‘Type 2 hypersensitivity granu- eases like Crohn disease, sarcoidosis, and primary bili-ary cirrhosis that are of unknown cause (Table 2) (16).lomas’’ may induce less organ damage and symptom-

atology than ‘‘type 1,’’ yet can still effectively reduce The latter presumably result from an aberrant height-ened, chronic immune response to benign environmentlocal and systemic spread of noxious factors from non-

invasive irritants. They are less effective at controlling factors or endogenous host proteins.Over their existence, granulomas evolve through sev-invasive microorganisms (11).

Recently described is a third type of hypersensitivity eral stages associated with sequential changes in cyto-kine expression (17–19). There is an elicitation phase,granuloma (12). This forms in response to the proto-

zoan Leishmania chagasi. These granulomas make a maintenance phase, and an involution phase. In theprimary granulomatous response, the granuloma niduspredominantly IL10 and IL6, and little or no IFN-g,

IL4, or IL5. IL10 is a cytokine synthesis inhibitory fac- embeds in tissue, causing tissue damage and local acti-vation of a variety of endogenous biochemical cascadestor (13). In the susceptible host, the organism finds

safety in the granuloma macrophages. It is probable such as the coagulation, complement, kinin, and angio-tensin systems. Products of tissue injury and the focalthat the parasite induces the granuloma macrophages

to produce large quantities of IL10 to evade destruction generation of various mediators such as IL1 (20–23),TNFa (24–27), MCP-1, and MIP-1a (28) attract andby the host’s immune system. Other microbial patho-

gens that reside in macrophages have developed sev- activate leukocytes. Although there are few T cells inforeign-body granulomas, T lymphocytes have a centraleral different defense mechanisms to survive in the

hostile intracellular environment (14, 15). role in induction, maintenance, and regulation of hy-persensitivity granulomas (2, 29). In hypersensitivityFactors that induce granulomas share several com-

mon physical attributes. They persist in host tissue, granulomas, host granuloma T cells recognize anti-genic determinants of the inciting nidus. Afterwards,cause tissue injury, and resist destruction from im-

mune attack. Many environmental factors induce gran- they proliferate and secrete cytokines that support andregulate the cellular immune response. In a primaryulomatous inflammation. Among the infectious agents

are mycobacteria, Leishmania, and Listeria. These or- hypersensitivity granulomatous reaction, the processof sensitization may take 10 days. Yet subsequent chal-ganisms persevere within granuloma macrophages. In

parasitic diseases such as schistosomiasis, ova lodge in lenges with the antigenic nidus result in sensitized Tcells rapidly entering the site of inflammation that, inhost tissue and release toxic and antigenic substances

that incite granulomas. The eggs remain extracellular, turn, promote an early, vigorous granulomatous re-sponse. ‘‘Primary-’’ and ‘‘secondary-type’’ hypersensi-protected from rapid destruction by the surrounding

eggshell. Minerals such as beryllium and zirconium be- tivity granulomas display both qualitative and quanti-tative differences in cytokine production. Secondary-have as antigens when complexed to proteins of the

susceptible host and cause granulomatous responses. hypersensitivity granulomatous reactions are thelesions seen most frequently in many chronic granulo-Moreover, other nonantigenic minerals like silicates,

mineral oil, and barium induce granulomas. Some drug matous disease states.Hypersensitivity granulomas are complex reactions

that use various cell-mediated and humoral immuneTABLE 1 mechanisms to control or destroy the inciting nidus.

Granulomas that form in response to various antigenicGranuloma Nomenclaturesubstances differ in their ultimate size, cell content,

Phases of granuloma development and expression of various effector immune mecha-Elicitation (induction) nisms. These are dictated by the nidus and to a greatMaintenance

extent the array of cytokines produced within the le-Involutionsions. Also, organs such as the lungs, liver, and intes-Granuloma classification

Foreign-body tines may respond differently to the same granulo-Hypersensitivity magenic substance due to regional host factors (30).

Type 1 Induction of Th1-type inflammation requires IL12 andType 2

IFN-g (31), whereas the shift to the Th2 pattern ofType 3inflammation may involve the expression of factorsOther ?

Hypersensitivity granuloma: Stages of response such as IL4 and IL10 during an early phase of thePrimary inflammation (32, 33). Mast cells activated through aSecondary variety of pathways may release IL4 that, in turn, mayFlorid

promote development of Th2-like lymphocytes. Nonan-Modulatedtigenic microbial oligosaccharides can induce expres-

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307IMPORTANCE OF GRANULOMA MODELS

sion of IL10 from B cells and possibly contribute to the flammatory factors, limiting granuloma growth and in-tensity. For instance, maintenance-phase granulomasdown-regulation of Th1 cells (34). In granulomatous

responses, the actual mechanisms of Th2 induction are make little IL1 or IL2, molecules important for initia-tion of vigorous inflammation (10, 38, 39). Yet they mayunknown.

In most hypersensitivity granulomas, TCR-ab// selectively continue production of other cytokines likeIFN-g, IL4, or IL5 that are critical for a variety ofCD4/ T lymphocytes predominate and are critically im-

portant for granuloma development (1, 35). Yet in some functions such as macrophage activation, IgE/IgG1 B-cell differentiation or eosinophil growth and develop-granulomas, TCR-ab//CD8/ T cells (36), NK cells (37)

and even TCR-gd/ T lymphocytes (29) clearly have im- ment, respectively (7, 10). Both T and non-T cells maymake some of these important cytokines (10, 40, 41). Inportant roles. Each, at times, can express a similar

cytokine profile, suggesting that they may have some schistosome granulomas, mast cells may be the majorsource of IL4 (unpublished observation). A variety ofoverlapping functions.

Within several days, granulomas may enter the overlapping cell–cell interactions and soluble factorslimit both the amount and the kinds expressed (41–maintenance phase. During this period, immunoregu-

latory mechanisms restrain production of some proin- 44). Important in this regard is the granuloma non-

TABLE 2

Some Conditions Associated with Granulomatous Inflammation of the Liver (L) and/or Intestines (I)

I. Infections Carbamazepine (L)Bacterial Chlorpromazine (L)

Atypical mycobacteria (L) Diphenylhydantoin (L)Brucellosis (L) Halothane (L)Cat scratch disease (L) Hydralazine (L)Leprosy (L) Methimazole (L)Tuberculosis (L, I) Methyldopa (L)Tularemia (L) Nitrofurantoin (L)Whipple disease (L) Phenylbutazone (L)Yersinia (I) Procainamide (L)

Chlamydial Quinidine (L)Sulfasalazine (L) Quinine (L)Lymphogranuloma venereum (L, I) Sulfasalazine (L)

Fungal Sulfonamides (L)Blastomycosis (L, I) Sulfonylurea (L)Coccidioidomycosis (L, I) III. MalignancyCryptococcosis (L) Hodgkin disease (L)Histoplasmosis (L, I) Non-Hodgkin lymphoma (L)Nocardiosis (L) IV. MetalsSystemic candidiasis (L) Barium (I)Trichosporosis (L) Beryllium (L)

Helminthic Copper (L)Ascariasis (L) V. VasculitisCapillariasis (L) Giant-cell arteritis (L)Schistosomiasis (L, I) Lupus erythematosus (L)Strongyloidiasis (L, I) Polyarteritis nodosa (L)Tongue worm (L) Polymyalgia rheumatica (L)Toxocariasis (Visceral larva migrans) (L) Rheumatoid arthritis (L)

Protozoan Wegener’s granulomatosis (L, I)Giardiasis (L) VI. MiscellaneousToxoplasmosis (L) Appendiceal granuloma (I)Visceral leishmaniasis (L) Bacille Calmette-Guerin immunotherapy (L)

Rickettsial Crohn disease (L, I)Q fever (L) Idiopathic granulomatous gastritis (I)

Spirochetal Idiopathic granulomatous hepatitis (L)Secondary syphilis (L, I) Immunodeficiency (L)

Viral Jejunoileal bypass surgery (L)Cytomegalovirus (L) Mineral oil (L, I)Mononucleosis (L) Primary biliary cirrhosis (L)

II. Drug hypersensitivity Sarcoidosis (L, I)Allopurinol (L) Silica (L)Amoxicillin-clavulanic (L)

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308 METWALI ET AL.

lymphoid, cellular element. This includes mature tis- vated leukocytes that are selected for deposition at thesite of injury. They use a variety of effector mecha-sue macrophages that can inhibit lymphocyte prolifera-

tion and both IL2 and IFN-g production (43–46). nisms, delicately balanced by many immunoregulatorycircuits. Granulomas can be readily isolated free fromThe granuloma enters the involution phase as de-

struction of the granuloma nidus slows the inflamma- contaminating parenchyma, which is a unique experi-mental advantage. It is easy to obtain large numberstory process. The lesions become less cellular and more

fibrotic. Eventually, the granuloma may resolve com- of dispersed granuloma inflammatory cells that areuseful for cellular, biochemical, and molecular analy-pletely or leave permanent scar tissue. Granulomas

form on a dynamic collagenous matrix that displaces ses. Using the various granuloma models, it is possibleto dissect the actual, local control mechanisms thatnormal organ parenchyma. Deposited are several colla-

gen types (47–49). Various cell types and many molecu- govern the granulomatous response. Granulomas usea variety of immunoregulatory networks and solublelar signals regulate collagen production, remodeling,

and resorption (50–52). Poorly defined are the mecha- cytokines common to many inflammatory states. Thus,knowledge of lesions learned studying granulomas isnisms that permit or restrict fibrosis. Predisposing ge-

netic factors allow some individuals to form scar tissue applicable to other types of inflammation. Experimen-tal granulomatosis has advanced our understanding ofand permanent organ damage more readily (53, 54).

In chronic infection, control of some pathogens does lymphokine regulation and gd T-cell function, as wellas of the importance of heat shock proteins. Also, theynot require a continuously strong granulomatous re-

sponse. Also, the chronic granulomatous inflammation have allowed identification of a variety of immunoregu-latory circuits not previously described. These pointscharacteristic of idiopathic diseases such as sarcoidosis

and Crohn disease cycles in intensity. The spontaneous are illustrated below.Animal models of granulomatous inflammationdecrease in the intensity of the host response to newly

deposited granulomagenic factors is termed ‘‘modula- helped prove that there is reciprocal regulation be-tween Th1 and Th2 cytokines (59, 60). Other importanttion.’’ As characterized in murine schistosomiasis man-

soni, modulation is controlled by dynamic interactions observations included the demonstration that IFN-gand IL12, through stimulation of IFN-g secretion, pre-between T-cell subsets (55, 56). It results in granulo-

mas that are less dynamic and smaller in size than vent Th2 pathway induction (31, 61). Moreover, granu-loma models helped probe the prominent functions ofthose developing in the florid inflammatory state. Yet

the capacity of the host to respond to other antigens cytokines such as TGF-b (62), IL5 (63), and IL10 (64).Granulomatous inflammations have shown that heatis intact. However, an ongoing Th2-like response may

impede full implementation of a Th1 response to an shock proteins can play a major role in host–pathogeninteractions. Heat shock proteins are ubiquitous,unrelated foreign antigen (57, 58). The immunoregula-

tory events required for induction of modulation are highly conserved sets of molecules. A variety of stressesinduce expression of these proteins by both the hostunknown. Modulation may limit symptomatology and

organ damage, thus benefiting the host who cannot and the invading pathogen. Heat shock proteins havebeen identified as major immunogens in granuloma-resolve his granulomatous response.

The frequently asked question is why should we tous diseases such as leprosy, tuberculosis, leishmania-sis, and schistosomiasis (65–68).bother to study granulomas? It is important to examine

granulomas because a variety of diseases trigger this Only a small subset of CD3/ T cells express a gdrather than the usual ab TCR. Yet gd T cells accumu-inflammatory process. It is the granulomatous re-

sponse that determines the outcome in mycobacterial late at sites of granulomatous inflammation. Althoughthe physiologic significance of gd T cells still remainsand many other microbial infections. Also, it is granulo-

matous inflammation that causes organ injury in idio- elusive, models of granulomatous inflammation areproviding insights into the mechanisms of gd T-cellpathic conditions such as sarcoidosis and Crohn dis-

ease. Thus, understanding the mechanisms that recruitment and activation as well as function (69–71).For instance, it has been shown that gd T cells reactenhance, diminish, or modulate granulomas could aid

in the treatment of many diseases. to mycobacterial heat shock protein and may play arole in early protective immunity to this pathogen (72,Moreover, many human granulomatous diseases in-

duced by infectious agents are transmissible to ani- 73). Also, in listeriosis, gd T cells may regulate theformation of the liver granulomas (29).mals. This allows sophisticated investigation of some

disease processes in a manner not possible using hu- The existence of a neurokine immunoregulatory cir-cuit within chronic inflammation was a unique discov-man subjects.

In addition, experimental models of granulomatous ery. Granuloma cells in murine schistosomiasis makevarious neurokines such as substance P, somatostatin,inflammation permit analysis of the basic immune

mechanisms involved in immunoregulation. Granulo- and vasoactive intestinal peptide. They work to selec-tively modulate cytokine expression (74). These obser-mas are chronic, focal inflammations composed of acti-

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309IMPORTANCE OF GRANULOMA MODELS

19. Wynn, T. A., Eltoum, I., Cheever, A. W., Lewis, F. A., Gause,vations have broad implications for the control of in-W. C., and Sher, A. (1993) J. Immunol. 151, 1430–1440.flammation at mucosal surfaces and other places where

20. Montreewasuwat, N., Curtis, J., and Turk, J. L. (1987) Cell.neurokines abound.Immunol. 104, 12–23.Moreover, these experimental models show that

21. Kasahara, K., Kobayashi, K., Shikama, Y., Yoneya, I., Soezima,studies using splenocytes or peripheral blood leuko- K., Ide, H., and Takahashi, T. (1988) Am. J. Pathol. 130, 629–cytes may not reveal the true, dominant immunoregu- 638.latory mechanisms employed at sites of active inflam- 22. Utsunomiya, I., Nagai, S., and Oh-Ishi, S. (1991) J. Immunol.mation. The leukocytes of spleens and blood are a 147, 1803–1809.mixture of cells in various stages of activation, many 23. Chensue, S. W., Bienkowski, M., Eessalu, T. E., Warmington,

K. S., Hershey, S. D., Lukacs, N. W., and Kunkel, S. L. (1993)of which are not destined to participate in a selectiveJ. Immunol. 151, 3654–3662.immune response. Also, granulomas are sustained in-

24. Kasahara, K., Kobayashi, K., Shikama, Y., Yoneya, I., Kaga, S.,flammatory reactions. They permit analysis of theHashimoto, M., Odagiri, T., Soejima, K., Ide, H., Takahashi, T.,unique features of chronic maintenance, as opposed toand Yoshida, T. (1989) Clin. Immunol. Immunopathol. 51, 419–

acute phase inflammation. 425.

25. Kindler, V., Sappino, A.-P., Grau, G. E., Piguet, P.-F., and Vas-salli, P. (1989) Cell 56, 731–740.

26. Joseph, A. L., and Boros, D. L. (1993) J. Immunol. 151, 5461–REFERENCES 5471.

27. Lukacs, N. W., Chensue, S. W., Strieter, R. M., Warmington, K.,and Kunkel, S. L. (1994) J. Immunol. 152, 5883–5889.1. McElrath, M. J., Murray, H. W., and Cohn, Z. A. (1988) J. Exp.

Med. 167, 1927–1937. 28. Lukacs, N. W., Chensue, S. W., Smith, R. E., Strieter, R. M.,Warmington, K., Wilke, C., and Kunkel, S. L. (1994) Am. J.2. Buchanan, R. D., Fine, D. P., and Colley, D. G. (1973) Am. J.Pathol. 144, 711–718.Pathol. 71, 207–214.

29. Mombaerts, P., Arnoldi, J., Russ, F., Tonegawa, S., and Kauf-3. Amiri, P., Locksley, R. M., Parslow, T. G., Sadick, M., Rector,mann, S. H. E. (1993) Nature 365, 53–56.E., Ritter, D., and McKerrow, J. H. (1992) Nature 356, 604–607.

30. Weinstock, J. V., and Boros, D. L. (1981) J. Immunol. 127, 1906–4. Flynn, J. L., Chan, J., Triebold, K. J., Dalton, D. K., Stewart,1909.T. A., and Bloom, B. R. (1993) J. Exp. Med. 178, 2249–2254.

31. Wynn, T. A., Eltoum, I., Oswald, I. P., Cheever, A. W., and Sher,5. Cooper, A. M., Dalton, D. K., Stewart, T. A., Griffin, J. P., Russell,A. (1994) J. Exp. Med. 179, 1551–1561.D. G., and Orme, I. M. (1993) J. Exp. Med. 178, 2243–2247.

32. D’Andrea, A., Aste-Amezaga, M., Valiante, N. M., Ma, X., Kubin,6. Squires, K. E., Schreiber, R. D., McElrath, M. J., Rubin, B. Y.,M., and Trinchieri, G. (1993) J. Exp. Med. 178, 1041–1048.Anderson, S. L., and Murray, H. W. (1989) J. Immunol. 143,

4244–4249. 33. Cheever, A. W., Williams, M. E., Wynn, T. A., Finkelman, F. D.,Seder, R. A., Cox, T. M., Hieny, S., Caspar, P., and Sher, A.7. Sacco, R. E., Jensen, R. J., Thoen, C. O., Sandor, M., Weinstock,(1994) J. Immunol. 153, 753–759.J., Lynch, R. G., and Dailey, M. O. Submitted for publication.

34. Velupillai, P., and Harn, D. A. (1994) Proc. Natl. Acad. Sci. USA8. Reiner, N. E., Ng, W., Wilson, C. B., McMaster, W. R., and Bur-91, 18–22.chett, S. K. (1990) J. Clin. Invest. 85, 1914–1924.

35. Mathew, R. C., Ragheb, S., and Boros, D. L. (1990) J. Immunol.9. Hirsch, C. S., Ellner, J. J., Russell, D. G., and Rich, E. A. (1994)144, 4356–4361.J. Immunol. 152, 743–753.

36. Chensue, S. W., Warmington, K. S., Hershey, S. D., Terebuh,10. Cook, G. A., Metwali, A., Blum, A., Mathew, R., and Weinstock,P. D., Othman, M., and Kunkel, S. L. (1993) J. Immunol. 151,J. V. (1993) Cell. Immunol. 152, 49–58.1391–1400.11. Pirmez, C., Yamamura, M., Uyemura, K., Paes-Oliveira, M.,

37. Scharton, T. M., and Scott, P. (1993) J. Exp. Med. 178, 567–577.Conceicao-Silva, F., and Modlin, R. L. (1993) J. Clin. Invest. 91,1390–1395. 38. Cook, G., Blum, A., Ballas, Z., and Weinstock, J. V. (1991) Neu-

roimmunology 33, 217–225.12. Wilson, M. E., Young, B. M., Sandor, M., Lynch, R. M., Blum,A. M., Metwali, A., Elliott, D., and Weinstock, J. V. Submitted 39. Metwali, A., Blum, A., Mathew, R., Sandor, M., Lynch, R. G.,for publication. and Weinstock, J. V. (1993) Cell. Immunol. 149, 11–23.

13. Moore, K. W., O’Garra, A. O., de Waal Malefyt, R., Vieira, P., 40. Williams, M. E., Kullberg, M. C., Barbieri, S., Caspar, P., Berzof-and Mosmann, T. R. (1993) Annu. Rev. Immunol. 11, 165–190. sky, J. A., Seder, R. A., and Sher, A. (1993) Eur. J. Immunol.

23, 1910–1916.14. Mauel, J. (1990) J. Leukocyte Biol. 47, 187–193.41. Metwali, M., Elliott, D., Blum, A., and Weinstock, J. V. Submit-15. Small, P. L. C., Ramakrishnan, L., and Falkow, S. (1994) Science

ted for publication.263, 637–639.42. Metwali, A., Elliott, D., Blum, A. M., and Weinstock, J. V. (1993)16. Neil, G. A., and Weinstock, J. V. (1991) in Textbook of Gastroen-

J. Immunol. 151, 7048–7056.terology, Chap. 105 (Yamada, T., Ed.), J. B. Lippincott, Philadel-phia, PA. 43. Metwali, A., Blum, A., Mathew, R., Sandor, M., Lynch, R. G.,

and Weinstock, J. V. (1993) Cell. Immunol. 149, 11–23.17. Chensue, S. W., Otterness, I. G., Higashi, G. I., Forsch, C. S.,and Kunkel, S. L. (1989) J. Immunol. 142, 1281–1286. 44. Mathew, R. C., Weinstock, J. V., Blum, A., and Metwali, A. (1992)

Gastroenterology 102, A660.18. Chensue, S. W., Terebuh, P. D., Warmington, K. S., Hershey,S. D., Evanoff, H. L., Kunkel, S. L., and Higashi, G. I. (1992) J. 45. Elliott, D. E., Ragheb, S., Wellhausen, S. R., and Boros, D. L.

(1990) Infect. Immun. 58, 1577–1583.Immunol. 148, 900–906.

/ 6706x$257s 04-19-96 06:59:01 metha AP: Methods

310 METWALI ET AL.

46. Flores Villanueva, P. O., Harris, T. S., Ricklan, D. E., and Sta- 61. Wang, Z.-E., Reiner, S. L., Zheng, S., Dalton, D. K., and Locksley,R. M. (1994) J. Exp. Med. 179, 1367–1371.decker, M. J. (1994) J. Immunol. 152, 1847–1855.

47. Biempica, L., Dunn, M. A., Kamel, I. A., Kamel, R., Hait, P. K., 62. Barral-Netto, M., Barral, A., Brownell, C. E., Skeiky, Y. A. W.,Fleischner, C., Biempica, S. L., Wu, C. H., and Rojkind, M. (1983) Ellingsworth, L. R., Twardzik, D. R., and Reed, S. G. (1992)Am. J. Trop. Med. Hyg. 32, 316–325. Science 257, 545–548.

48. Grimaud, J. A., Boros, D. L., Takiya, C., Mathew, R. C., and 63. Sher, A., Coffman, R. L., Hieny, S., Scott, P., and Cheever,Emonard, H. (1987) Am. J. Trop. Med. Hyg. 37, 332–345. A. W. (1990) Proc. Natl. Acad. Sci. USA 87, 61–65.

49. Meneza, S. E., Olds, G. R., Kresina, T. F., and Mahmoud, A. A. 64. Denis, M., and Ghadirian, E. (1993) J. Immunol. 151, 5425–F. (1989) Hepatology 9, 50–56. 5430.

50. Olds, G. R., Meneza, S. E., Mahmoud, A. A. F., and Kresina, 65. Rey-Ladino, J. A., and Reiner, N. E. (1993) Infect. Immun. 61,T. F. (1989) J. Immunol. 142, 3605–3611. 3265–3272.

51. Manthey, C. L., Allen, J. B., Ellingsworth, L. R., and Wahl, 66. McKenzie, K. R., Adams, E., Britton, W. J., Garsia, R. J., andS. M. (1990) Growth Factors 4, 17–26. Basten, A. (1991) J. Immunol. 147, 312–319.

52. Greenwel, P., Wyler, D. J., Rojkind, M., and Prakash, S. (1993) 67. Shinnick, T. M. (1991) Curr. Top. Microbiol. Immunol. 167, 145–Infect. Immun. 61, 3985–3987. 160.

53. Cheever, A. W., Dunn, M. A., Dean, D. A., and Duvall, R. H. 68. Hedstrom, R., Culpepper, J., Harrison, R. A., Agaian, N., and(1983) Am. J. Trop. Med. Hyg. 32, 1364–1369. Newport, G. (1987) J. Exp. Med. 165, 1430–1435.

54. Cheever, A. W. (1987) Am. J. Trop. Med. Hyg. 37, 85–97. 69. Sandor, M., Lynch, R. G., Cook, G. A., Weinstock, J. V., Sperling,A., and Bluestone, J. A. (1994) J. Immunol., in press.55. Yamashita, T., and Boros, D. L. (1990) J. Immunol. 145, 724–

731. 70. Uyemura, K., Klotz, J., Pirmez, C., Ohmen, J., Wang, X.-H., Ho,C., Hoffman, W. L., and Modlin, R. L. (1992) J. Immunol. 148,56. Fidel, P. L., Jr., and Boros, D. L. (1990) J. Immunol. 145, 1257–1205–1211.1264.

71. Rosat, J.-P., MacDonald, H. R., and Louis, J. A. (1993) J. Immu-57. Kullberg, M. C., Pearce, E. J., Hieny, S. E., Sher, A., and Berzof-nol. 150, 550–555.sky, J. A. (1992) J. Immunol. 148, 3264–3270.

72. Pechhold, K., Wesch, D., Schondelmaier, S., and Kabelitz, D.58. Pearlman, E., Kazura, J. W., Hazlett, F. E., and Boom, W. H.(1994) J. Immunol. 152, 4984–4992.(1993) J. Immunol. 151, 4857–4864.

73. Inoue, T., Yoshikai, Y., Matsuzaki, G., and Nomoto, K. (1991) J.59. Heinzel, F. P., Sadick, M. D., Holaday, B. J., Coffman, R. L., andImmunol. 146, 2754–2762.Locksley, R. M. (1989) J. Exp. Med. 169, 59–72.

60. Pearce, E. J., Caspar, P., Grzych, J.-M., Lewis, F. A., and Sher, 74. Blum, A. M., Metwali, A., Mathew, R. C., Elliott, D., and Wein-stock, J. V. (1993) J. Immunol. 151, 6994–7004.A. (1991) J. Exp. Med. 173, 159–166.

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