Response in Distant Lymph Nodes of Mice to Infection in the

Transcription

1 INFECTION AND IMMUNITY, Apr. 1980, p /80/ /05$02.00/0 Vol. 28, No. 1 Response in Distant Lymph Nodes of Mice to Infection in the Hind Footpad with Mycobacterium marinum NATAN MOR, IRVING LUTSKY, AND LOUIS LEVY* Department of Comparative Medicine, Hebrew University-Hadassah Medical School, Jerusalem, Israel In an attempt to demonstrate the importance of the popliteal lymph node in limiting the progress of infection with Mycobacterium marinum in the hind footpads of C57BL mice, such infections were studied in mice subjected to popliteal or popliteal and inguinal adenectomies. In the absence of the popliteal node, the footpad infection was only slightly enhanced compared with infections of sham-operated control mice; the inguinal node was found to be greatly enlarged and appeared to have substituted for the absent popliteal node. In the absence of both popliteal and inguinal nodes, the disease process in the footpads was again only slightly enhanced, and the axillary node appeared to have enlarged greatly and to have functionally replaced the missing, more proximate nodes. In additional experiments, mice subjected to adenectomy only on one side and injected in that hind footpad with phytohemagglutinin or India ink demonstrated hypertrophy or deposition of carbon particles in the more distant node only on the side of the injection. Thus, there appear to be rather direct functional connections among popliteal, inguinal, and axillary nodes that do not depend on blood circulation. The popliteal lymph node, which drains the hind footpads of mice (1), becomes an active site of lymphocytic proliferation during infection in the footpads with Mycobacterium marinum (Mor, Lutsky, and Levy, unpublished data). The number of lymphocytes in the paracortical area of the popliteal node increased greatly during the first 2 weeks of infection. If it may be assumed that specifically committed T-lymphocytes produced in the popliteal node are essential for limiting the disease process that follows in of a hind footpad with M. marinum, one might anticipate that excision of the lymph node would permit enhanced multiplication and delay killing of the organisms. We report here the results of experiments designed to study the effects of removal of the draining lymph node on infection of mouse footpads with M. marinum. (The results were taken from a Ph.D. thesis submitted by N.M. to the Hebrew University- Hadassah Medical School.) MATERIALS AND METHODS Locally bred C57BL/6 mice were employed. The culture of M. marinum was obtained from the Leprosy Research Unit, Public Health Service Hospital, San Francisco, Calif. To prepare a standard inoculum of M. marinum, a broth culture (Dubos broth base with Dubos serum supplement [Difco Laboratories, Detroit, Mich.]) was inoculated and shaken for 3 days at 32 C. The concentration of the organisms, determined directly on a stained preparation (5), was 8.12 x 105 ± 1.75 x 105 (mean ± Student's to.95 X standard deviation) acid-fast bacilli per ml. Dimethyl sulfoxide was added to a final concentration of 10% (vol/vol), the suspension was distributed among a number of tubes, and the portions of bacterial suspension were frozen by a modification of the method of Colston and Hilson (3). The number of colony-forming units in a thawed portion of the suspension was determined to be 3.1 x 105 ± 1.1 x 105 per ml, representing a loss by freezing of approximately 60%. At the time of in, the frozen standard inoculum was thawed by immersion in a 37 C water bath and diluted with 0.1% (vol/vol) bovine serum albumin in Hanks balanced salt solution to yield a concentration of 5 x 103 M. marinum cells per 0.03 ml. Mice were inoculated in one or both hind footpads by the method described for Mycobacterium leprae by Shepard (7). M. marinum cells were harvested, usually from the pooled tissues of four infected footpads, and the acidfast bacilli were counted directly on Reich counting slides (Bellco Glass Co., Vineland, N.J.) after staining with a standard acid-fast stain (8). The number of colony-forming units was determined by plating suitably diluted suspensions in duplicate on Middlebrook 7H10 agar with Middlebrook albumin Dubos complex enrichment (Difco). Plates were incubated at 32 C for 7 to 10 days. Footpad thickness was measured by using a dial gauge caliper (The Dyer Co., Inc., Lancaster, Pa.) (6). Tissue specimens were fixed in Carnoy or Bouin fluid and processed for histopathological examination. Sections 3 to 4,um thick were stained with hematoxylin and eosin and by the acid-fast stain of Fite et al. (5). To prepare single-cell suspensions from lymph 225

2 226 MOR, LUTSKY, AND LEVY nodes, the nodes were suspended in Hanks balanced salt solution and pressed first through a fine mesh metal sieve and then through hypodermic needles of successively smaller gauge. RESULTS At 1 week after surgery, popliteal adenectomized, popliteal and inguinal adenectomized, and sham-operated mice were inoculated with approximately 5,000 viable M. marinum cells into both hind footpads. As Tables 1 and 2 show, foot thickening occurred to the same extent in popliteal adenectomized and sham-operated mice. However, multiplication of M. marinum, in terms of the numbers of acid-fast bacilli and colony-forming units recovered from the footpads, appears to have continued somewhat longer and to a somewhat greater maximum in popliteal adenectomized mice compared with sham-operated mice. The weights of the inguinal nodes, the numbers of lymphocytes that could be expressed from the nodes, and the numbers of M. marinum cells recovered from the inguinal nodes (in terms of colony-forming units) in popliteal adenectomized mice were much greater TABLE 1. than the corresponding measurements for shamoperated mice. Thus, in the absence of the popliteal nodes, the inguinal nodes became sites of active lymphocytic proliferation. In a second experiment (Tables 3 and 4) as in the first, no difference in foot thickness between popliteal and inguinal adenectomized mice and sham-operated mice was noted. However, multiplication of M. marinum in the footpads of popliteal and inguinal adenectomized mice appeared to have been enhanced. Compared with the axillary nodes of the sham-operated mice, those of the popliteal and inguinal adenectomized mice became greatly enlarged (Fig. 1), and increases in node weight, number of lymphocytes that could be expressed, and colony-forming units occurred. Thus, the axillary nodes appeared to have substituted for the absent popliteal and inguinal nodes. Histopathological examination of the enlarged inguinal nodes obtained from popliteal adenectomized mice and of the enlarged axillary nodes obtained from popliteal and inguinal adenectomized mice revealed changes similar to those noted in the popliteal nodes of sham-operated Responses in the footpads ofpopliteal adenectomized C57BL mice to infection in both hind footpads with M. marinum Time after in- Footpad thickness (mm)p No. of acid-fast bacilli per foot- CFU/footpad (x105)b (days) Adenectomized Sham-operated Adenectomized Sham-operated Adenectomized Sham-operated ± 0.10C 1.48 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.1 NMd 0.54 ± 0.11 NM ± ± ± ± ± ± 0.01 a Measurements were performed on 10 feet. b Measurements were performed on pooled tissues of four footpads. CFU, Colony-forming units. d NM, Not measured. TABLE 2. Responses in the inguinal lymph nodes ofpopliteal adenectomized C57BL mice to infection in both hind footpads with M. marinum Wt(mg/node)'No. of lymphocytes per node CFU/node (XlIO:Y Time after in- W (mg/node) (x10o)a (days) Adenectom- Sham-oper- Adenectomized Sham-operated Adenectomized Sham-operated ized ated 0 4.8b c ± ± ± ± ± ± ± ± ± ± ± ± ± ameasurements were performed on pooled tissues of four lymph nodes. CFU, Colony-forming units. 'Mean ± Student's to.s5 X standard deviation. INFECT. IMMUN.

3 VOL. 28, 1980 TABLE 3. DISTANT NODES IN M. MARINUM INFECTION OF MICE 227 Responses in the footpads ofpopliteal and inguinal adenectomized C57BL mice to infection in both hind footpads with M. marinum Time after in- Footpad thickness (mm)' No. of acid-fast bacilli per foot- CFU/footpad (X105)b pad (XlO5)b (days) Adenectomized Sham-operated Adenectomized Sham-operated Adenectomized Sham-operated ± 0.10C 1.50 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.03 a Measurements were performed on 10 feet. b Measurements were performed on pooled tissues of four footpads. CFU, Colony-forming units. TABLE 4. Responses in the axillary nodes ofpopliteal and inguinal adenectomized C57BL mice to infection in both hind footpads with M. marinum Wt(mg/node)'No. of lymphocytes per node CFU/node (XI03), Time after in- Wt1 (mg/node)0 (x17)a CFU/nodea (days) Adenectom- Sham-opersized ated Adenectomized Sham-operated Adenectomized Sham-operated ± 0.02c 0.21 ± ± ± ± 0.20 < ± ± ± 0.50 < ± ± ± 0.18 < ± ± ± 0.19 <0.003 Measurements were performed on pooled tissues of four lymph nodes. CFU, Colony-forming units. b Mean. C Mean ± Student's to.95 x standard deviation. ;rn FIG. 1. Enlarged axillary lymph node of a popliteal and inguinal adenectomized mouse sacrificed 21 days after in of the hind footpad with M. marinum. The arrow indicates the axillary node. mice and in the popliteal nodes of unoperated mice. The paracortical area was densely populated with small lymphocytes, and collections of epithelioid macrophages were seen throughout the lymph nodes (Fig. 2). Occasionally, massive proliferation of macrophages was found to have obliterated the subcapsular and medullary lymph sinuses. Acid-fast bacilli were observed in the cytoplasm of the macrophages. In the inguinal nodes of the sham-operated mice, limited proliferation of lymphocytes and accumulation of macrophages were observed; no pathological changes were noted in the axillary nodes of these animals. The granulomatous proliferation of the macrophages observed in the footpads of the popliteal adenectomized and popliteal and inguinal adenectomized mice was similar to that in the sham-operated mice. Additional experiments were performed, in which phytohemagglutinin (Difco) and India ink were used, seeking evidence to confirm the presence of functional connections among popliteal, inguinal, and axillary lymph nodes. In these experiments, popliteal or popliteal and inguinal adenectomies were performed on the right sides, and the left sides served as controls. In one experiment, 0.5 mg of phytohemagglutinin was injected into the- right hind footpads of mice adenectomized 7 days earlier, and animals were sacrificed immediately or 4 or 6 h after injection.

4 228 MOR, LUTSKY, AND LEVY Sii-w4 b y ; A -j l As Tables 5 and 6 show, comparisons of lymph _4##g^/i Jf*node weights and lymphocyte counts revealed ;'1"ubf5<4^nB~>2** Idly- W s A) important differences between the lymph nodes pi01 > Jw' of the right and left sides of the injected animals. *.X ",h*tg*, ;t,-,j>.,':* *..* In every instance, the changes in response to phytohemagglutinin injection were limited to the side of the injection, and in the adenectom- V_2,;.;*i _.,. a ized animals, the enlargement of the more distant node was both remarkable and limited to t ft a) -. * the side of the injection. ;"j"s- SetO In a second experiment, India ink was injected., into the right hind footpads of two mice that ;gi:2>4~ ga # fir A, ; ' 0, i had been subjected to popliteal and inguinal * am i"t t 1, adenectomies on the right side 7 days earlier; Jgit.X5*li ;'( gf A, ' the animals were sacrificed 7 h after injection. us-,f* ^ ;!4t lp 9t*# t~ttw < Carbon particles were found in the subcapsular 'P Elf 4 "f<,,st i~s X.. and perinodular sinusoidal cells of the right ax- *t.x* >0tk '-}4.) a-;-42,<** illary nodes, whereas no particles were found in wring;<,,-it.,^ -";,d&&4,:ss ' Gus the left axillary nodes. 7~~~~~~~ ~ ~ <toa. ^ z *it DISCUSSION Ot#.i.. '., Because of the intensive lymphocytic prolift, - a Do ' eration in the paracortical area of the popliteal FIG. 2. Accumulation of macrophages in the axil- lymph node that accompanied infection in hind lary node 14 days after in of the hind foot- footpads with M. marinum (Mor, Lutsky, and Ipad of a popliteal and inguinal adenectomized Levy, unpublished data), it was anticipated that mouse. Hematoxylin and eosin stain. x180. removal of the popliteal node would remove the TABLE 5. Responses of the inguinal nodes of right popliteal adenectomized C57BL mice to injection in the right hind footpads with 0.5 mg ofphytohemagglutinin Wt (mg/node)' No. of lymphocytes per node (x106)" Time Adenectom- Nra after Normal sized Normal Adenectomized injection Popliteal Inguinal Inguinal Popliteal Inguinal Inguinal Right Left Right Left Right Left Right Left Right Left Right Left 0 10b ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.3 Measurements were performed on pooled tissues of three lymph nodes. TABLE 6. Responses of the axillary nodes of right popliteal and inguinal adenectomized C57BL mice to injection in the right hind footpads with 0.5 mg ofphytohemagglutinin Time Wt (mg/node)' No. of lymphocytes per node (x106), after in- Normal Adenectomized Normal Adenectomized jection (h) Right Left Right Left Right Left Right Left 0 3.5b ± 0.1c 2.9 ± ± ± ± ± ± ± ± ± ± ± 0.2 ameasurements were performed on pooled tissues of three lymph nodes. e Mean ± Student's to.s5 X standard deviation. INFECT. IMMUN.

5 VOL. 28, 1980 DISTANT NODES IN M. MARINUM INFECTION OF MICE 229 primary source of specifically sensitized T-lymphocytes, resulting in enhancement of the disease process. However, excision of the popliteal or the popliteal and inguinal lymph nodes before infection with M. marinum resulted only in minimal enhancement of the disease process, suggesting that the adenectomies did not prevent the arrival of antigen in effective concentrations at alternative sites at which sensitized T-lymphocytes might be produced. The locations of the lymph nodes of mice have been described, but the areas drained by the individual nodes have not been delineated (4). One study has shown that injection of India ink into the hind footpads of mice stained primarily the popliteal and sciatic nodes, with the inguinal nodes stained only to a very slight degree (1). However, the distribution of antigen beyond the popliteal node has not been studied thoroughly in an infectious process. It has been reported that most of the organisms that leave a footpad during infection reach the popliteal node; the inguinal node has been regarded as not directly connected to the popliteal node, receiving bacilli by way of the circulation (2), whereas the sciatic and lumbar nodes were thought to be directly connected (1). The demonstration here of functional substitution of the inguinal node for the popliteal node of the same side and of the axillary node for the popliteal and inguinal nodes of the same side suggested that these more distant nodes are not ordinarily exposed to effective concentrations of antigen. After excision of the more proximate node, antigen was distributed to the more distant node not primarily by way of blood circulation, but rather directly, as if by lymphatic channels. This was confirmed by the ipsilateral response to the injection of phytohemagglutinin and by the appearance of carbon particles only in the ipsilateral axillary node after injections of India ink into one hind footpad of animals doubly adenectomized on that side. Thus, in the absence of the popliteal node, the organisms appeared to have been distributed primarily to the inguinal node, and in the absence of both popliteal and inguinal nodes, they were distributed to the axillary node. The results of these experiments suggest that contact between lymphoid tissue and antigen from locally injected M. marinum takes place by way of lymphatic channels and that blood circulation does not play a major role in this process. In M. marinum infections of hind footpads of the C57BL mice, the popliteal, inguinal, and axillary lymph nodes appear to operate as a series of filters draining the footpads, with the lymph reaching the axillary nodes almost sterile; this may be the mechanism by which dissemination of the infection is prevented. LITERATURE CITED 1. Carter, P. B., and F. M. Collins The route of enteric infection in normal mice. J. Exp. Med. 139: Closs, Experimental murine leprosy. Growth of Mycobacterium lepraemurium in C3H and C57BL mice after footpad in. Infect. Immun. 12: Colston, M. J., and G. R. F. Hilson The effect of freezing and storage in liquid nitrogen on the viability and growth of Mycobacterium leprae. J. Med. Microbiol. 12: Dunn, T. B Normal and pathological anatomy of the reticular tissue in laboratory mice, with a classification and discussion of neoplasia. J. Natl. Cancer Inst. 14: Fite, G. L., P. S. Cambre, and M. H. Turner Procedure for demonstrating lepra bacilli in paraffin sections. Arch. Pathol. 43: Ng, H., P. L. Jacobsen, and L. Levy Analogy of Mycobacterium marinum disease to Mycobacterium leprae infection in footpads of mice. Infect. Immun. 8: Shepard, C. C The experimental disease that follows the injection of human leprosy bacilli into footpads of mice. J. Exp. Med. 112: Shepard, C. C., and D. H. McRae A method for counting acid-fast bacteria. Int. J. Lepr. 36:78-82.