Role of Complement in Chemotaxis: Study of a Localized Infection

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1 INFECTION AND IMMUNITY, July 198, p /8/7-8/5$2./ Vol. 29, No. 1 Role of Complement in Chemotaxis: Study of a Localized Infection DENISE M. WILSON, DOUGLAS J. ORMROD, AND THOMAS E. MILLER* Department ofmedicine, Auckland Hospital, Auckland, New Zealand A model of Escherichia coli-induced pyelonephritis was used to study the effect of complement depletion in an organ-specific, nonimmunological inflammatory lesion in rats. In this model of a local infection, which can be considered to be a nonspecific inflammatory stimulus, the depletion of complement by the administration of a purified cobra venom factor did not alter the course of the disease. There were minor differences when the results from complement-depleted and normocomplementemic animals were compared, but the composition of the inflammatory infiltrate was not greatly altered. Therefore, the presence of C3 and a functional complement system are relatively unimportant factors in determining the characteristics of the inflammatory response in a localized infection-induced lesion. One of the postulated roles of the complement system in immunity is to mobilize immune effector cells into an inflammatory lesion. This activity is believed to be mediated through the components of activated complement, C3a and C5a; although these components may be regarded as having the potential to amplify the cellular component of the inflammatory response, their contribution to the morphogenesis of the inflammatory response in a localized bacterial infection has not been investigated. Although it is generally accepted that a prompt inflammatory response is an important element of the host's response to infection, the generation of a cellular response is not always associated with the eradication of infection. The model of experimentally induced renal infection used in this study is an example of an infection that persists despite an intense local inflammatory response. A reproducible model of Escherichia coli-induced pyelonephritis was used to study the role of complement in an organ-specific localized inflammatory lesion in rats. Animals were depleted of complement by the administration of purified cobra venom factor (). The course of the disease was studied in complement-depleted and normocomplementemic animals and, since has been shown to abrogate C3 (3), we could evaluate the importance of this component as a factor contributing to the intensity, composition, and nature of the inflammatory lesion. Our results show that in this model of a local organ infection, complement depletion does not alter the course of the disease. Although there were minor differences when results from complement-depeleted and replete animals were compared, the overall composition of the inflammatory infiltrate was not greatly altered. Bacterial replication and gross pathology were likewise unaffected. Therefore, it appears that certain complement components, although potentially capable of affecting cellular mobilization, play a minor role in determining the characteristics of the inflammatory response. MATERIALS AND METHODS Animals. Male and female DA rats from an inbred strain were used. Selection of organism. A strain of E. coli 8 isolated in a previous study (12) was used as the challenging organism. Production of renal infection. Pyelonephritis was induced by directly inoculating E. coli 8 into the surgically exposed kidney with a Hamilton syringe (The Hamilton Co., Whittier, Calif.). The culture was adjusted with saline so that logo 4.2 viable E. coli were administered in 1!d. Animals for examination 24 and 48 h after challenge were inoculated with the logo 4.2 cells; the other animals were inoculated with the logo 3.5 cells. Details of the model have been previously described (13).. was isolated from Naja naja venom (Sigma Chemical Co., St. Louis, Mo.) as previously described (1). The fractions containing anticomplementary activity after diethylaminoethyl chromatography were concentrated and further fractionated on a Sephadex G-2 column (Pharmacia Ltd., Uppsala, Sweden). Active fractions were again pooled, concentrated, and then stored in equal portions at -7'C. Decomplementation procedure. Animals were decomplemented by the intraperitoneal administration of 1,g of purified as a divided dose with 8 h between administration. Determination of total serum hemolytic complement (CH5M) and C3 determination. Details of the methods used have been described previously (12). 8 Downloaded from on May 12, 218 by guest

2 VOL. 29, 198 The 5% hemolysis endpoint procedure developed by Taliaferro and Taliaferro (15) and described in detail by Campbell et al. (2) was used to determine the hemolytic complement activity. Quantitative analyses of C3 were carried out by rocket electrophoresis. All serum samples were stored at -7'C. Quantitative histological evaluation. Quantitative assments of both the degree of renal damage and the composition of the cellular infiltrate were made. A kidney profile was reproduced on paper by using a stencil, and the extent of the renal lesions was estimated by eye and then transferred to the kidney profile by shading in the appropriate areas. The paper profile was cut out, weighed, and then reweighed after the shaded area, representing the damaged tissue, had been removed. The area of the section involved in the lesion was expressed as a percentage of the total area of the section. Quantitative basement of the cellular components in the pyelonephritic lesion was done with the aid of a graticule placed in the eyepiece of the microscope. The number of cells per unit area in the lesion was determined and differentiated into polymorphs, lymphocytes, and mononuclear cells, and nuclear remnants from the renal epithelial and tubular cells. Hematology. Blood was collected from the tail vein, using a 1-ml syringe with a 26-gauge needle, and immediately expressed into an evacuated tube containing anticoagulant (ethylenediaminetetraacetic acid). The blood was diluted in an isotonic diluent and processed in a Coulter Counter, model S. The total leukocyte count, hemoglobin level, and erythrocyte indices were determined. A blood film was examined for differential leukocyte count and erythrocyte morphology. Bacteriology. Nutrient agar pour plates of serial 1-fold dilutions of kidney homogenate were made to obtain a bacterial count per gram of wet tissue. Quantitative evaluation of gross pathological changes. Quantitation of the gross renal pathology was made on a to 1 scale, with each unit of the scale representing 1% of the surface of the kidney. Statistical evaluation. Statistical evaluation of results was carried out on a PDP11 computer, usin the Wilcoxon rank sum test for nonparametric data. RESULTS Decomplementation procedure. was administered by intraperitoneal injection in two doses 8 h apart. Two and five days after the administration of, the CH5 and C3 levels in the serum were below 2% or normal. Eight days after administration, the CH5 had returned to normal and the C3 level had risen to 14% of normal (Fig. 1). Effect of decomplementation on renal bacteriology and gross pathology. Forty animals were selected and divided into two groups. One group was decomplemented with, and the other group served as a control. The next day the left kidneys of all animals were challenged with E. coli 8. -ru COMPLEMENT IN CHEMOTAXIS C3 conc. o---o CH5 1 2 infection i7-=--n days after treatment FIG. 1. Effect of administration on the CH5 and C3 concentration in the rat. Purified (.2 ml) was administered intraperitoneally as a divided dose with 8 h between administrations. Renal infection was induced 12 h after the second dose. The points shown are the mean percentage of baseline values from six animals. Animals from each group were sacrificed 24, 48, 72, and 96 h after the induction of infection, and the left (pyelonephritic) kidney was removed. Animals to be sacrificed 24 and 48 h after challenge were infected with logo 4.2 viable E. coli; those examined 72 and 96 h after challenge were infected with logo 3.5 cells. The gross appearance of the excised kidneys was scored before they were homogenized in saline for estimation of bacterial numbers. Minor differences were apparent when the gross pathological and bacteriological changes found in the treated and untreated groups were compared. These differences were confined to the early phase of the disease, however, and by 3 days the groups were indistinguishable by these criteria (Fig. 2). Histopathological features of the inflammatory infiltrate. In another group, 4 animals were decomplemented, infected, and sacrificed as described in the preceding section. The infected kidneys from both -treated and untreated animals were processed and examined for histopathological changes. Decomplementation did not have a marked effect on the inflammatory response, but some changes of interest were found. Twenty-four hours after challenge, a marginally significant increase (P <.5) in the percentage ofpolymorphonuclear leukocytes (PMNL) per unit area of lesion in the treated rats could be demonstrated, whereas at 96 h the percentage of PMNL in the treated animals was significantly reduced (P <.1). The other significant result was that the degree of damage in the -treated animals / 9 Downloaded from on May 12, 218 by guest

3 1 WILSON, ORMROD, AND MILLER con. A it [ 24 hr ] v 6cA con. [48 hr ] 13 * a En 2 1 * *-con c Iba cterolo g~y. con [72 hr ] gross pathology A con. con. I * *- * a so con. so CFTV [92 hrj" * l I-- con. con, FIG. 2. Effect of decomplementation on the bacteriological and gross pathological parameters in experimentally induced renal infection. Quantitation of the gross pathology was made on a to 1 scale, with each unit representing 1% of the surface area of the kidney. was increased 48 h after challenge (P <.1) (Table 1). Hemotological parameters. A significant increase in leukocyte numbers in the treated rats could be 'demonstrated soon after decomplementation but 9 days after the leukocyte count had returned to normal. The differential leukocyte count of both the control and -treated groups remained unaltered over the 9-day period (Fig. 3). A decrease in erythrocyte numbers demonstrated in both groups was attributed to the effects of sequential blood sampling carried out over a short interval. Other erythrocyte characteristics were unaffected. INFECT. IMMUN. DISCUSSION In these experiments we investigated in vivo the role of the complement system, and specifically C3, on the development of the cellular infiltrate in a localized inflammatory lesion. Decomplementation of laboratory animals was done by using purified, and the effect of this manipulation on the mobilization of inflammatory cells into a localized E. coli-induced infectious lesion in the kidney was determined. Complement depletion did not alter the rate of sequestration of inflammatory cells into the renal lesion or the composition of the cellular infiltrate. The ability of to decomplement laboratory animals was established in preliminary experiments. was found to be effective in reducing the total hemolytic complement activity to below detectable levels (<.5% of normal) and in maintaining C3 levels of less than 3% of normal for up to 6 days. The effect of on peripheral blood leukocytes and other hematological factors was examined, and a significant but general increase in leukocyte numbers was found 2 and 6 days after administration. treatment did not affect erythrocyte parameters. These results closely follow those reported by Cochrane et al. (3). Histopathological examination of the infected kidney from -treated animals 48 h after infection showed a small but significant increase in the area of tissue affected by scarring when compared with untreated animals. An increase in PMNL was found at 24 h in the complementdepleted animals; however, a slight decrease in PMNL was evident at 96 h in the -treated group. Several related studies have been carried out in an effort to elucidate the role of complementderived chemotactic factors in the inflammatory response. Jungi and McGregor (9) found that C3-related factors were not essential for the expression of delayed-type hypersensitivity or protection against a challenge with Listeria monocytogenes. In a further series of experiments, however, Lawrence and Schell (1) claimed that T cells and complement components, independently or cooperatively, did in fact play a role in immunity to L. monocytogenes. Complement depletion has been shown to affect the pathology of necrotic skin lesions, and in experiments using a Staphylococcus aureus-induced lesion, it was shown that the ultimate dimension of the lesion was proportional to the number of polymorphs in the early infiltrate (4). Tissue enzymes released by granulocytic cells are potentially tissue destructive and have been implicated in the generation of infectious lesions (11). It was our hope that a reduction of the inflammatory infiltrate would lessen the degree of pathological damage to the pyelonephritic kidney, but as we showed, the absence of complement activity did not affect the mobilization of inflammatory cells into the kidney, and it was therefore not possible to test this hypothesis. Complement or complement-associated factors may, however, be important determinants Downloaded from on May 12, 218 by guest

4 VOL. 29, 198 TABLE 1. COMPLEMENT IN CHEMOTAXIS 11 Histopathologicalfeatures ofpyelonephritic kidneys, of area affected Polymorphs/unit Lymphocytes and Nuclear rem- Day Group No. of animalst area unit area DayGroup No. of~~~ oarafecd aea mononuclear cells/ nants/unit area Mean Signifi- Mean Signifi- Mean Signifi- Mean Signifi- (SD) cance (SD) cance (SD) chance (SD) cance Day 1 Control (54) (1.5) (41.8) R (26) (.76) (15.4) NS.5 NS NS Day 2 Control (1.9) (2.7) (4.4) (23.2) R (2.74) (2.) (1.2) (36.6).1 NS NS NS Day 3 Control (3.7) (17.1) (1.6) (15.8) R (4.2) (17.) (8.4) (15.6) NS NS NS NS Day 4 Control (6.) (13.) (16.4) (18.5) R (4.2) (9.32) (18.6) (19.2) NS.1 NS NS SD, Standard deviation. b Level of significance when results from -treated animals ( R) are compared with those from control animals, using the Wilcoxon rank sum test for nonparametrc data. NS, no significant difference ] 8 2 W B Cx 19/l 1l *-' R *-* control % PMNL l /% monocytes % -lymphocytes 16~6 61, 2 Z days after R, FIG. 3. Total kukocyte count and differential eu. kocyte count of normal and complemnent-depleted ( R) animals monitored over a 1-day period in the eradication of pathogens. Jarvinen and Dalmasso (8) demonstrated that C3 depletion led to a reduced rate of parasite elimination, but it is not clear whether this was related to a reduced inflammatory response. Gross et al., in a series of experiments in mice, studied the role of complement in early pulmonary clearance of organisms frequently associated with human pneumonia (7). Phagocytic clearance of two of the four organisms used was unaffected, whereas clearance of the other two was significantly depressed. The degree of depression was small, however, and it is doubtful that decomplementation would have had any effect on the ultimate course of the disease. Many studies have demonstrated the chemotactic properties of C3a and C5a in vitro (5, 6, 14). These components can be cleaved from their precursors, C3 and C5, either by sequential activation of the complement cascade or by enzymes extrinsic to the complement system. Enzymes with this activity include trypsin, plasmin, and thrombin, as well as certain tissue and bacterial proteases that can utilize C3 and C5 as subtrates but do not sustain sequential component interactions. In contrast to the in vitro studies, the mechanisms by which C3a and C5a are produced in vivo have not been elucidated. Downloaded from on May 12, 218 by guest

5 12 WILSON, ORMROD, AND MILLER Activation of the complement system by microorganisms, however, is not necessary to ensure that complement-cleaving enzymes are present in inflammatory lesions; rather, they are derived from the cellular components of the lesion. Thus, a complement activating microorganism in a local lesion is not necessarily a prerequisite for complement dependent chemotaxis. The local inflammatory lesion in the kidney can be regarded as a nonspecific inflammatory stimulus, and although there may be an immunological component, there is nothing to suggest that the infectious stimulus equates to the "immunological" injury involved in the pathogenesis of a number of renal lesions involving the glon eruli. In summary, complement may have a chemotactic role in lesions induced by certain bacterial, immunological, and nonimmunological processes. Many biological components display chemotactic characteristics, however, and the consensus opinion is that the role of complement-derived chemotaxis is a minor one. Spontaneous random migration or attraction by such factors as bacterial metabolites or products of cellular necrosis should be considered as more likely mechanisms. LITERATURE CED 1. Ballow, IL, and C. G. Cochrane Two anticomplementary factors in cobra venom: hemolysis of guinea-pig erythrocytes by one of them. J. Immunol. 13: Campbell, D. IL, J. S. Garvey, N. E. Cremer, and D. IL Sussdorf p In Methods in immunology. W. A. Benjamin Inc., New York. 3. Cochrane, C. G., IL J. Muller-Eberhard, and D. S. Aitkin Depletion of plasam complement in vivo INFECT. IMMUN. by a protein of cobra venom: its effect on various immunologic reactions. J. Immunol. 15: Easmon, C. S. F., and A. A. Glynn Comparison of subcutaneous and intraperitoneal staphylococcal infections in normal and complement-deficient mice. Infect. Immun. 13: Fernandez, H. N., P. M. Henson, A. Otani, and T. E. Hugli Chemotactic response to human C3a and C5a anaphylatoxins. I. Evaluation of C3a and C5a leucotaxis in vitro and under simulated in vivo conditions. J. Immunol. 12: Goldstein, I. M Endogenous regulation of complement (C5)-derived chemotactic activity: fine-tuning of inflammation. J. Lab. Clin. Med. 93: Gross, G. N., S. R. Rehm, and A. K. Pierce The effect of complement depletion on lung clearance of bacteria. J. Clin. Invest. 62: Jarvinen, J. A., and A. P. Dalmasso Trypanosoma musculi infections in normocomplementemic, C5- deficient and C3-depleted mice. Infect. Immun. 16: Jungi, T. W. and D. D. McGregor Role of complement in the expression of delayed-type hypersensitivity in rats: studies with cobra venom factor. Infect. Immune. 23: Lawrence, D. A., and R. F. Schell Susceptibility of C5-deficient mice to listeriosis: modulation by concanavalin A. Cell. Immunol. 39: Leffell, AL S., and J. K. Spitznagel Intracellular and extracellular degranulation of human polymorphonuclear azurophil and specific granules induced by immune complexes. Infect. Immun. 1: Miller, T. E., S. Phillips, and L J. Simpson Complement-mediated mechanisms in renal infection. II. Effect of decomplementation. Clin. Exp. Immunol. 33: Miller, T. E., and K. B. Robinson Experimental pyelonephritis: a new method for inducing pyelonephritis in the rat. J. Infect. Dis. 127: Brother, K Leucocyte mobilising factor: a new biological activity derived from the third component of complement. Eur. J. Immunol. 2: Taliaferro, W. IL, and L G. Taliaferro The dynamics of hemolysin formation in intact and splenectomized rabbits. J. Infect. Dis. 87: Downloaded from on May 12, 218 by guest