American Journal ofPathology, Vol. 136, No. 1, January 1990 Copyright © American Association ofPathologists

Subcutaneous Perfusion of Tumor Necrosis Factor Induces Local Proliferation of Fibroblasts, Capillaries, and Epidermal Cells, or Massive Tissue Necrosis Pierre F. Piguet, Georges E. Grau, and Pierre Vassalli From the Department ofPathology, Centre Medical

Universitaire, Geneva, Switzerland

Mouse recombinant tumor necrosis factor (TNF) (or its solvent alone as a control) was administered subcutaneously to mice by a cannula connected to an osmotic minipump. Perfusion at a rate of 35 ng/hr for seven days induced the formation of a tissue mass composed mainly of fibroblasts, collagen, and capillaries. Necrosis (apoptosis) of isolated fibroblasts was observed. Polymorphonuclear leukocytes were abundant after three to four days ofperfusion but were absent later. The covering epidermis showed a hyperplastic reaction associated with necrosis of isolated keratinocytes. Perfusion at a rate of 170 ng/hr led, afterfour to five days, to a massive necrosis. Necrosis was completely prevented by rabbit anti- TNFIgG but not by anti-LPS IgG, irradiation, or administration of indomethacin. (Am JPathol 1990, 136:103-1 10)

Tumor necrosis factor-alpha/cachectin (TNF) is a cytokine produced by macrophages stimulated by agents such as lipopolysaccharides (LPS). Its various properties have been the subject of recent reviews.12 Production of TNF also is increased during the immune response, and particularly during immunopathologic reactions.3-5 In a study of the graft-versus-host reaction (GVHR), we have observed that the cutaneous lesions could be prevented by anti-TNF antibody,4 thus suggesting that TNF might cause pathologic reactions in the skin. The local physiopathologic effects resulting from administration of TNF have been explored thus far by single or repeated injections in the skin, which resulted in an inflammatory reaction or an hemorrhagic necrosis resembling the Shwartzman reaction when TNF was injected together with LPS or interleukin-1.6 9 In the present study,

the lesions produced by a continuous hypodermic TNF perfusion were explored by light and electron microscopy. The perfusion was delivered over three to seven days by a cannula connected to an osmotic minipump and the investigation was focused on the site of cytokine delivery, a procedure that minimizes the nonspecific reactions produced by surgical trauma or the pump itself. Depending on the mouse strain, the dose of TNF, and the duration of perfusion, a wide variety of lesions were observed, including notably keratinocyte hyperplasia, fibroblast accumulation with collagen deposition, neo-angiogenesis and, with high doses, complete necrosis.

Materials and Methods Mice C57BL/10 (B1i0), Balb/c, CBA/Ca mice were purchased initially from Olac 1976 Ltd, Bicester, Oxon, U.K., and were bred in our animal house in conventional conditions. DBA/2 and C3HHeJ were obtained from Jackson Laboratories, Bar Harbor, ME. Irradiation was performed in a 135Ce irradiator, with 500 rad delivered in four minutes.

TNF Perfusion Esherichia coli mouse recombinant TNF-alpha (mrTNF) was provided by B. Allet, Glaxo IMB, Geneva, Switzerland. The specific activity evaluated in a TNF assay using the L 929 fibroblast cell line10 was 3 X 1 07 units/mg, one unit being defined as the activity required to lyse 50% of the cells. Contamination with endotoxin was below 1 ng/ Lgg of TNF, as determined by the Limulus amoebocyte Supported by Grant No. 3.550.87 from the Swiss National Science Foundation. Accepted for publication August 30, 1989. Address reprint requests to Pierre F. Piguet, Departement de Pathologie, 1 rue Michel, Servet, 1211 Geneva, Switzerland.

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lysate assay. The cytokine, diluted in Hank's balanced salt solution, supplemented with penicillin G (103 U/ml and streptomycin sulfate 103 ,g/ml), was delivered by an osmotic minipump (2001 or 2002, Alza, Palo Alto, CA) at a flow rate of 1 Al/hr. The pump was implanted subcutaneously in the groin region, and the outlet was connected to a catheter of about 15 mm in length, the cross-shaped extremity of which was implanted in the subcutaneous tissue of the flank. Control mice received an osmotic pump containing the solvent.

Drugs Indomethacin (Sigma Chemical Co., St Louis, MO) was added to the drinking water at a final dilution of 107 M.

Antibodies Rabbit anti-mrTNF antibodies were raised as previously described.3-5 The anti-TNF neutralizing activity, assayed in the TNF assay,10 was detectable at a 1/250 dilution of the 1 mg/ml IgG solution. Anti-LPS antibodies were raised in rabbits by injections of E. coli LPS (055:B5, Difco Laboratories, Detroit, Ml) in Freund's complete adjuvant. One milligram per milliliter of the anti-LPS IgG fraction had a binding titer of 1/280 in an enzyme-linked immunosorbent assay (ELISA). Recipient mice were passively immunized with 1 mg of the IgG fraction, previously ultra-centrifuged, and injected intraperitoneally at the beginning of the perfusion. One milligram of the anti-LPS IgG did protect galactosamine-sensitized mice against the lethal effects of an injection of LPS.'1

Evaluation of the Collagen Deposition by the Dosage of Hydroxyproline A square of about 2 cm in border, centered around the extremity of the cannula, was excised. The dermis and epidermis were separated and the hypodermic tissue was processed to evaluate its hydroxyproline content12 as an evaluation of the collagen.

Light and Electron Microscopy The area around the cannula was fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer. For light microscopy, the tissues were embedded in methyl-metacrylate and sections were stained with hematoxylin and eosin (H & E). For light and electron microscopy (EM), tissues were embedded in epon and sections were stained with toluidine

blue. Ultrathin sections were examined with a Philips 400 electron microscope.

Results The results obtained with various protocols of TNF perfusion are summarized in Table 1. Perfusion of TNF, but not of the solvent alone, at a dose of > 170 ng/hr led after four to five days to a massive necrosis (Figure 1). At a dose of 35 ng/hr, a tissue neoformation slowly grew around the site of infusion, reaching approximately 2 cm in diameter on day 7 (Figure 2A to D). The tissue was composed predominantly of fibroblasts, collagen fibrils of various sizes, capillaries, and small blood vessels (Figure 2F, 3A, B). The walls of blood vessels and connective tissue were infiltrated by polymorphonuclear leukocytes after three to four days of perfusion (Figure 2E), whereas these cells were rare or absent later (Figure 2F). Fibroblasts were most often round with a richly developed endoplasmic reticulum (Figure 2F, 3A). Some showed a margination of the heterochromatin or an eccentric nucleus (Figure 3A). Fibroblasts with large apoptotic vesicles were observed occasionally (Figure 2 G, H). After seven days of TNF infusion at 35 ng/hr, the evaluation of the quantity of collagen around the site of perfusion showed a 2.5-fold increase over the control (Table 2). Epidermal reactions were observed occasionally during TNF perfusion at 35 ng/hr (probably when the tip of the catheter was close enough to the epidermis), but were constant in some strains after three to four days of TNF perfusion at 170 ng/hr, ie, before complete necrosis. The epidermis above the site of TNF perfusion, showed a hyperplastic reaction, as seen by: 1) thickening of the epidermis, which became five to ten cells elevated, compared with one to three for the control (Figure 2B, D); 2) an increase (about 20-fold) of the number of mitoses per microscopic field (Figure 3D); 3) anisocytosis and vacuolization of the keratinocytes (Figure 3D); and 4) necrosis of isolated keratinocytes (Figure 3D). As mentioned above, perfusion at a rate of > 170 ng/ hr led to a complete necrosis in all of the strains tested (Table 1). After four to five days of perfusion, the necrosis appeared macroscopically as a brown induration of the skin (Figure 1 B), which evolved toward ulceration on day 7 (Figure 1 C). On histologic sections, the tissue around the site of perfusion showed a coagulation necrosis with little infiltration by inflammatory cells (Figure 3C, E). Necrosis did involve the epidermis, dermis, hypodermis, and the thoracic wall, and reached the ribs, which showed signs of active resorption or complete necrosis (not shown). Vascular lesions, such as parietal thrombosis or hemorrhages, were observed occasionally on the border of the necrosis. Tissue obtained after two to four days of perfu-

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Table 1. Continuous Hypodermic Perfusion of TNF

TNF

Mouse strain B10

Treatment None None None

Balb/c

None None None None None None None None rx 1 * anti TNF anti LPS Indometh.

CBA CBA CBA C3H/HeJ C3H/HeJ DBA/2 B10 B10 B10 B10

Fibrosist angiogenesis

(ng/hr) 0 35 170 35 170 35 170 850§ 0 170 170 170 170 170 170

Epidermal

hyperplasia

Necrosist

+ + + ++

-(0/12) - (0/12) ++ (12/12) - (4/4) + (4/6) -(0/5) ++ (4/12)

+ ++ + +

+ ++

+ ++

+ +

+ +

-(0/3) ++ (4/4) + (4/4) ++ (6/6)

-(0/6)

++ (4/4) ++ (6/6)

Summary of the morphological (macroscopic and histological) findings observed after seven days of perfusion. * Mice were treated as described in methods. Irradiation (rx) was administered three days before the implantation of the pump. Antibody was injected before the implantation of the pump. t Fibroblastic and angiogenic reaction were scored as follows: -, not seen; +, foci detected microscopically; ++, formation of a tissue mass > 2 cm in

diameter. t The necrosis were scored macroscopically and histologically as follows: -, not seen, +, area of necrosis in dermis and epidermis, 1 cm in diameter. Number of animals with necrosis/number of animals examined. § Mice dead after 2 to 3 days of perfusion.

sion and examined by light or electron microscopy showed evidences of cellular damage, such as extensive vacuolization (Figure 3D) or various stages of cellular disintegration; these were observed in keratinocytes, endothelial cells, fibroblasts, and adipocytes. Damage of endothelial cells was associated occasionally with parietal fibrin

deposition but was not associated with massive thrombosis or extensive extravasation of red blood cells. The intensity of these reactions varied considerably between different mouse strains (Table 1). The majority of the strain tested, such as the C57B1/1 0, Balb/c, the LPS resistant C3HHeJ, and the complement-deficient DBA/2,

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Figure 1. C3HHeJ mice after seven days ofperfusion ofsaline (A) or TNF, 200 ng/hrforfour (B) or seven days (C). The site ofcytokine delivery is indicated by a thin arrow, the approximative position of the pump by a wide arrow.

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Figure 2. A-E: Perfusion with solvent(A,B) or mrTNF, 170 ng/hr (CDE), day 4 A-C: An intense cellular infiltration in the dermis (d), hypodermis (h), striated muscle (m) as well as an hyperplastic reaction of the epidermis are evident in C, but not in A. The site of cytokine delivery is indicated by a star (H&E, X 10). B&D; the epidermis is normal in B while in D the epidermis is elevated, with an increase of the size ofall epidermal layers (H&E, X40). E: An hypodermic blood vessel is seen with elevated endothelial cells (e) and a wall infiltrated bypolymorphonuclear leukocytes, some of these marked by arrows (TB, X400). F-H: Perfusion with TNF35 ng/hr, day 7 F: Numerous round shapedfibroblasts as well as capillaries (c) are seen, (TB, X 160). G, H: Fibroblasts, surrounded by collagen fibrils (f), are containing large apoptotic vesicles (EM, X5800 and X 6000).

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Table 2. Increase of Collagen Deposition by Infusion of TNF Mice

Perfusion

(ng/hr) B10

Solvent

mrTNF, 35

Hydroxyproline

(Mg) 4911 122 (6)*

Mean (±sd) of the values observed with 8 Bi 0 mice. * Different from the solvent: P > 0.01.

susceptible to both proliferative (epidermal and fibroangiogenic) and necrotic reactions. In addition, several C57BL1 0 bearing various H-2 loci showed responses identical to the B10 (not shown). In the CBA strain, the proliferative responses were minimal and the necroses were inconstant; in two experiments, no reaction was observed whereas in another, performed with another batch of CBA mice, four of five developed an extensive necrosis. To explore the pathogenesis of the necrosis, various treatments were attempted (Table 1). TNF-induced necrosis was prevented completely by injection of rabbit antimouse TNF antibody, whereas nonimmune or anti-LPS rabbit IgG had no effect. The necrosis was neither prevented nor markedly decreased by irradiation given three days before the beginning of the perfusion; this treatment led to a medullary aplasia with a drastic reduction in the number of circulating polymorphonuclear leukocytes, from 6 X 1 06/ml to