Cellular basis of host defence in pyelonephritis. III. Deletion of individual components

Hosts were depleted of individual cellular components to determine the effects of these manipulations on cellular defence mechanisms in acute and chronic pyelonephritis. T-lymphocytes were found to have little or no involvement in host protection but cyclosporin A administration had a dramatic effect on the gross pathology and bacteriological status of experimentally induced pyelonephritis. This change represented a major depression of host defence status. Cyclosporin A also activated resolved lesions in chronic pyelonephritis, associated with an increase in bacterial numbers. Administration of antineutrophil serum also led to a 1000-fold increase in bacterial numbers in the acute phase but had little effect on the host-parasite balance in chronic pyelonephritis. Macrophage blockade, on the other hand, did not affect the course of either acute or chronic infection. These studies have provided additional information on the immunobiology of experimental pyelonephritis and have focussed attention on the role of neutrophils, and an unidentified mechanism, affected by cyclosporin A, in host defence to renal infection.     

Cellular basis of host defence in pyelonephritis. II. Acute infection.

We have investigated the cellular basis of host defence mechanisms in experimental pyelonephritis. Cellular components of the host defence system were depleted using cyclophosphamide, methylprednisolone or radiation. Depletion of cellular competence did not affect the course of infection during the first 16 h after challenge with Escherichia coli, but after 96 h up to a 1000-fold increase in bacterial numbers in the kidneys of cytodepleted animals was demonstrable. When quantitative aspects of the relationship between cellular competence and host defence were studied, it was found that severe depletion of cellular components was necessary before host defence mechanisms were adversely affected. Thus while cellular mechanisms are quantitatively adequate and contribute to host defence in pyelonephritis they have little impact on the immediate post-infection phase. Non-cellular factors however, do limit bacterial proliferation in the acute phase and may be important determinants in the biology of pyelonephritis.     

Cellular basis of host defence in pyelonephritis. II. Acute infection

We have investigated the cellular basis of host defence mechanisms in experimental pyelonephritis. Cellular components of the host defence system were depleted using cyclophosphamide, methylprednisolone or radiation. Depletion of cellular competence did not affect the course of infection during the first 16 h after challenge with Escherichia coli, but after 96 h up to a 1000-fold increase in bacterial numbers in the kidneys of cytodepleted animals was demonstrable. When quantitative aspects of the relationship between cellular competence and host defence were studied, it was found that severe depletion of cellular components was necessary before host defence mechanisms were adversely affected. Thus while cellular mechanisms are quantitatively adequate and contribute to host defence in pyelonephritis they have little impact on the immediate post-infection phase. Non-cellular factors however, do limit bacterial proliferation in the acute phase and may be important determinants in the biology of pyelonephritis.     

Cellular basis of host defence in pyelonephritis. I. Chronic infection.

Infection persists for long periods in chronic pyelonephritis, but the cellular basis of the host-parasite relationship is poorly understood. We have obtained quantitative data on the relationship between the pathogen (E. coli) and cellular defence mechanisms. Depletion of cellular components was carried out using whole body irradiation, methylprednisolone, cyclophosphamide or carrageenan and silica particles. A system of administering cyclophosphamide and methylprednisolone through the use of a slow release carrier, as well as graded doses of irradiation, was then developed to allow the controlled reduction of cellular competence. Quantitative studies in a host with chronic pyelonephritis and normal cellular defence reserves showed that severe depletion of granulocytic cells is necessary before host defence mechanisms are adversely affected. This finding conflicts with the observation that microorganisms survive and persist in the kidney for extended periods. Additionally, noncellular factors may also limit bacterial growth.     

Cellular basis of host defence in pyelonephritis. I. Chronic infection

Infection persists for long periods in chronic pyelonephritis, but the cellular basis of the host-parasite relationship is poorly understood. We have obtained quantitative data on the relationship between the pathogen (E. coli) and cellular defence mechanisms. Depletion of cellular components was carried out using whole body irradiation, methylprednisolone, cyclophosphamide or carrageenan and silica particles. A system of administering cyclophosphamide and methylprednisolone through the use of a slow release carrier, as well as graded doses of irradiation, was then developed to allow the controlled reduction of cellular competence. Quantitative studies in a host with chronic pyelonephritis and normal cellular defence reserves showed that severe depletion of granulocytic cells is necessary before host defence mechanisms are adversely affected. This finding conflicts with the observation that microorganisms survive and persist in the kidney for extended periods. Additionally, noncellular factors may also limit bacterial growth.     

Exacerbation of experimental pyelonephritis by cyclosporin A

An athymic rat strain lacking functional T cells was used to assess the role of cell-mediated immunity (CMI) in host defence against renal infection. CMI was ruled out as a relevant host defence component, but when cyclosporin A (CsA) was administered to athymic animals, renal infection was exacerbated. CsA is thought to affect T lymphocyte function and, in the absence of a target cell, cellular defences in the athymic animal were not expected to be compromised by CsA. An effect on non-cellular defence mechanisms was therefore considered but our studies did not support this explanation--rather they indicated a depression of either cellular defences or of a specific cellular component. The present experiments have provided additional information on the relationship between CsA administration and the depression of host defence mechanisms but further studies will be necessary to identify the components affected.     

Mechanisms of recrudescence of Mycobacterium bovis BCG infection in mice.

The capacity of various immunosuppressive agents to cause a recrudescence of the replication of Mycobacterium bovis BCG in the spleens of chronically infected mice was investigated. The actions of three corticosteroid preparations, cyclosporin A, and anti-T-cell subset monoclonal antibodies were compared. Treatment of mice with hydrocortisone acetate, which depressed the number of splenic lymphocytes and suppressed T-cell responses, most effectively exacerbated the stationary BCG counts, at 4 to 6 months after infection. The magnitude of reactivation was more pronounced in innately resistant CBA/Ca mice than in the susceptible C57BL/6 strain of mice. Splenic bacterial counts were also amplified by anti-L3T4 antibody when the antibody was injected at the chronic phase, whereas cyclosporin A had an effect only during the initial 6 weeks after BCG infection. Cultures of spleen cells from chronically infected mice showed a significant increase in the numbers of viable BCG recovered after 7 days of incubation in the presence of dexamethasone but not with cyclosporin A. The observed differences between the tested immunosuppressive agents indicate that the stationary bacterial counts during chronic BCG infection are maintained by discrete T-cell actions on the infected macrophages.     

Host defence mechanisms in the bladder. II. Disruption of the layer of mucus.

The urinary bladder wall is lined by a layer of mucus which is believed to provide an important barrier to bacterial invasion of the urinary tract. Abnormal function of this protective layer could therefore be a factor predisposing the host to urinary tract infection (UTI). This study investigated the contribution of the bladder mucus to host defence in both acute and chronic lower UTI, using a non-obstructive animal model of infection which reproduces many features of the disease in man. The ultrastructural appearance of the infected bladder mucosa was assessed in tissue in which both the layer of mucus and bacterial glycocalyces were stabilized prior to examination by scanning and transmission electron microscopy. The protective role of the mucus layer was determined by disrupting the layer immediately prior to bacterial challenge. Both ultrastructural and bacteriological analyses have shown that infection was increased in those animals where the mucus barrier was disrupted.     

Host defence mechanisms in the bladder. II. Disruption of the layer of mucus

The urinary bladder wall is lined by a layer of mucus which is believed to provide an important barrier to bacterial invasion of the urinary tract. Abnormal function of this protective layer could therefore be a factor predisposing the host to urinary tract infection (UTI). This study investigated the contribution of the bladder mucus to host defence in both acute and chronic lower UTI, using a non-obstructive animal model of infection which reproduces many features of the disease in man. The ultrastructural appearance of the infected bladder mucosa was assessed in tissue in which both the layer of mucus and bacterial glycocalyces were stabilized prior to examination by scanning and transmission electron microscopy. The protective role of the mucus layer was determined by disrupting the layer immediately prior to bacterial challenge. Both ultrastructural and bacteriological analyses have shown that infection was increased in those animals where the mucus barrier was disrupted.     

Immunological enhancement in the pathogenesis of pyelonephritis.

The role of the immune response in pyelonephritis was investigated by manipulation of the host's immune capacity using the immunosuppressive drugs 6-mercaptopurine, cyclophosphamide and thiamphenicol. Treatment with 6-mercaptopurine depressed the humoral immune response but did not have an adverse effect on the course of renal infection. Thiamphenicol administration prevented the development of pathological lesions but this was due to the anti-bacterial activity of thiamphenicol and not to its immunosuppressive activity. Pyelonephritic animals treated with cyclophosphamide did not produce anti-bacterial antibody. Despite this, cyclophosphamide-treated animals were able to eliminate organisms more readily from the infected kidney than untreated animals with a normal humoral immune response. We believe that blocking of the phenomenon of immunological enhancement explains these unexpected results and that the immune response to renal infection may have an immunoenhancing role protecting the bacterial cell from otherwise effective host defence mechanisms.     

Relationship between neutrophil-mediated oxidative injury during acute experimental pyelonephritis and chronic renal scarring.

Previous experiments with rats have suggested that pyelonephritic scarring after acute ascending Escherichia coli pyelonephritis partly results from excessive polymorphonuclear leukocyte (PMN) infiltration and activation in the kidney parenchyma. We have studied the role of PMN oxidative metabolism in generating tissue injury during acute pyelonephritis. Rats with acute pyelonephritis were treated with dapsone (25 mg/kg twice daily for 3 days), a compound known to prevent PMN oxidant damage. In vitro, levels of dapsone easily achieved in vivo inhibited myeloperoxidase (MPO)-mediated reactions involving the oxidation of halides to reactive cytotoxic hypohalites (such as MPO-mediated iodination and luminol-enhanced chemiluminescence). In contrast, dapsone had no effect on superoxide production, lysosomal enzyme release, or bacterial killing by activated PMN. In vivo, dapsone treatment had no significant effect on acute pyelonephritis with respect to (i) bacterial counts, (ii) inflammatory swelling, and (iii) PMN infiltration. However, dapsone-treated animals sacrificed 2 months after acute pyelonephritis had a 65% reduction of renal scars when compared with controls. Since dapsone had no antibacterial effect, this protection is compatible with the hypothesis that dapsone prevented oxidant-generated tissue injury due to the extracellular release of the MPO system by activated PMN during acute suppurative pyelonephritis.     

Effects of cyclosporin A on experimental infection with Listeria monocytogenes.

The effects of cyclosporin A on primary and secondary infection of mice with Listeria monocytogenes was studied both at the microbiological and the histomorphological level. This drug, when given in a dose of 100 mg/kg/day, was found to inhibit the development of protective immunity after primary infection as well as the expression of acquired immunity to challenge infection as determined by counting of bacterial numbers in the spleen. The manifestation of delayed type hypersensitivity was also impaired. When the cellular immune system was functionally intact, the formation of granulomas composed of macrophages and lymphocytes enabled the animals to overcome the Listeria infection. In mice treated with cyclosporin A protective granulomatous reaction during secondary infection did not occur. Instead numerous necropurulent lesions developed in the reticuloendothelial organs, such as spleen and liver, of animals unable to control the lethal infection.     

Virulence of Escherichia coli in relation to host factors in women with symptomatic urinary tract infection.

The relationship between bacterial characteristics and the severity of urinary tract infection in adults has not been clarified. In this study, Escherichia coli strains (n = 178) were prospectively collected from women with community-acquired urinary tract infection. The isolates were identified by O:K:H serotype and characterized for adherence, hemolysin production, and serum bactericidal resistance. The patients had acute pyelonephritis with or without complicating factors and acute cystitis. Nine serotypes (O1:K1:H7, O1:K1:H-, O2:K1:H-, O4:K12:H1, O7:K1:H-, O9:K34:H-, O16:K1:H6, O16:K1:H-, and O75:K5:H-) comprised 65% of the strains in uncomplicated pyelonephritis, but were significantly less often encountered in complicated pyelonephritis or cystitis. Adherence was the single property most characteristic of the pyelonephritogenic clones. Adhesins specifically recognizing Gal alpha 1----4Gal beta-containing receptors occurred in 80% of strains in uncomplicated pyelonephritis, in 50% of strains in complicated infections, and in 37% of cystitis strains. Hemolysin production and serum resistance did not correlate with any disease pattern. Advanced age did not seem to reduce the selection of virulent E. coli to cause pyelonephritis. These results demonstrate in women a relationship between E. coli virulence and the severity of urinary tract infection analogous to that previously observed in pediatric populations and also illustrate the balance between host resistance and bacterial virulence in the urinary tract.     

Replication dynamics of Mycobacterium tuberculosis in chronically infected mice

The dynamics of host-pathogen interactions have important implications for the design of new antimicrobial agents to treat chronic infections such as tuberculosis (TB), which is notoriously refractory to conventional drug therapy. In the mouse model of TB, an acute phase of exponential bacterial growth in the lungs is followed by a chronic phase characterized by relatively stable numbers of bacteria. This equilibrium could be static, with little ongoing replication, or dynamic, with continuous bacterial multiplication balanced by bacterial killing. A static model predicts a close correspondence between "viable counts" (live bacteria) and "total counts" (live plus dead bacteria) in the lungs over time. A dynamic model predicts the divergence of total counts and viable counts over time due to the accumulation of dead bacteria. Here, viable counts are defined as bacterial CFU enumerated by plating lung homogenates; total counts are defined as bacterial chromosome equivalents (CEQ) enumerated by using quantitative real-time PCR. We show that the viable and total bacterial counts in the lungs of chronically infected mice do not diverge over time. Rapid degradation of dead bacteria is unlikely to account for the stability of bacterial CEQ numbers in the lungs over time, because treatment of mice with isoniazid for 8 weeks led to a marked reduction in the number of CFU without reducing the number of CEQ. These observations support the hypothesis that the stable number of bacterial CFU in the lungs during chronic infection represents a static equilibrium between host and pathogen.     

Bacterial and host determinants of renal scarring

This review summarizes recent work examining the interaction between host and parasite in recurrent urinary tract infection (UTI) and renal scarring. Virulence in uropathogenic E. coli has been defined by the severity of acute disease. Isolates from patients with acute pyelonephritic strains differ from those causing asymptomatic bacteriuria by multiple traits which contribute to virulence, and which are coexpressed in a non-random manner. The single marker most characteristic for the pyelonephritogenic clones is bacterial adherence to uroepithelial cells binding specifically to the disaccaride Gal alpha 1-4 Gal beta within the globoseries of glycolipids. The notion that the most severe consequence of acute pyelonephritis, i.e. renal scarring, was caused by the most virulent clones, was contradicted by comparison of pyelonephritic strains isolated from children with and without scarring. The virulent clones were significantly less frequent in patients with renal scarring (22%) than in patients with recurrent pyelonephritis not developing renal scars (62%). In view of the unexpected inverse association of bacterial virulence with renal scarring lack of Gal alpha 1-4 Gal beta binding capacity of E. coli strains was found to predict the risk for renal scarring among boys with first-time acute pyelonephritis. Vesicoureteric reflux (VUR) is widely accepted as a host determinant of susceptibility to pyelonephritis and renal scarring. In our study the frequency of renal scarring was 57% among girls with VUR as compared to 8% of those without. The reflux alone did however, not explain the selection of bacteria of low virulence. Individuals prone to UTI and renal scarring were found to be a genetically selected subgroup of the general population. A correlation between P1 blood group phenotype and susceptibility to UTI and between blood group non-secretor state and renal scarring was found. The mechanisms behind these relationships need to be defined. The bacterial and host parameters combined indicate that host parameters are essential for the tendency to develop renal scarring after acute pyelonephritis.     

Value of serum C-reactive protein measurement in the management of bone marrow transplant recipients. Part II: Late post-transplant period.

Seventeen bone marrow recipients transplanted for acute leukaemia (8), chronic leukaemia (1), severe aplastic anaemia (3), and various inborn errors of metabolism (5) had 22 episodes of documented infection in the late (greater than 3 months) post-transplant period. Serum C-reactive protein concentrations were considerably increased in patients with bacterial infections, but not in those with viral or fungal infections. Serum C-reactive protein values were normal in 20 patients transplanted for acute leukaemia (12), chronic leukaemia (1), severe aplastic anaemia (2), and various inborn errors of metabolism (5) who had active chronic graft versus host disease but no evidence of infection. These findings indicate that serum C-reactive protein concentrations are useful in the diagnosis and monitoring of bacterial infections even in the presence of chronic graft versus host disease.     

Influence of agglutinating antibody in experimental cryptococcal meningitis.

A model for chronic Cryptococcus neoformans meningitis in corticosteroid-treated rabbits was used to determine the influence of pre-formed agglutinating antibody to cryptococcal polysaccharide on the progress of this infection. Immunized rabbits developed serum agglutinating antibody with a geometric mean titre of 1:32, but none was detected in cerebrospinal fluid. Prior immunization did not enhance immunity to infection, had no effect on the number of viable cryptococci in cerebrospinal fluid, and did not prevent dissemination outside the central nervous system. Future investigations in this field should focus on cellular rather than humoral defence mechanisms.     

Mast cells: the forgotten cells of renal fibrosis

BACKGROUND/AIMS: Mast cells, when activated, secrete a large number of fibrogenic factors and have been implicated in the development of fibrotic conditions of the liver, lung, and skin. There is evidence that renal fibrosis is closely linked with a chronic inflammatory cell infiltrate within the interstitium, but a potential role for mast cells in this process has yet to be defined. Therefore, the numbers of mast cells in normal and fibrotic kidneys with various pathologies were investigated. METHODS: Mast cells were quantified in renal transplants showing acute and chronic rejection and cyclosporin toxicity, kidneys removed for chronic pyelonephritis, and renal biopsies from patients with IgA nephropathy, membranous nephropathy, and diabetic nephropathy. Mast cells were stained using two methods: acid toluidine blue detected less than 30% of the mast cells revealed by immunohistochemistry for mast cell tryptase. RESULTS: Mast cells were scarce or absent in normal kidney (median, 1.6 mast cells/mm2) but numerous throughout the cortex and medulla in all specimens that showed fibrosis. They were almost entirely confined to the renal interstitium. Mast cells were present in large numbers in biopsies from patients with membranous nephropathy (median, 21.7 mast cells/mm2) and diabetic nephropathy (median, 29.2 mast cells/mm2), which were selected on the basis of showing chronic injury. In 24 unselected IgA nephropathy biopsies there was a close correlation between numbers of mast cells and the extent of interstitial fibrosis (r = 0.771; p < 0.0001). In renal transplant biopsies, mast cells were associated with allograft fibrosis in chronic rejection (median, 27.1 mast cells/mm2) and chronic cyclosporin toxicity (median, 10.6 mast cells/mm2) but not acute rejection (median, 2.7 mast cells/mm2) or acute cyclosporin toxicity (median, 2.0 mast cells/mm2). There was no detectable increase in mast cell numbers during acute rejection in those transplants that subsequently progressed to chronic rejection. In some biopsies the mast cells were largely intact, but in most cases some or all were degranulated. CONCLUSIONS: An increased number of mast cells is a consistent feature of renal fibrosis, whatever the underlying pathology, and the number of mast cells correlates with the extent of interstitial fibrosis. This suggests that mast cells might play a pathogenetic role in the fibrotic process.     

Systemic humoral immunity to non-typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHi) is a major cause of respiratory but rarely systemic infection. The host defence to this bacterium has not been well defined in patients with chronic airway infection. The aim of this study was to assess the effect of humoral immunity in host defence to NTHi. Responses were measured in control and bronchiectasis subjects who had recurrent bronchial infection. Antibody and complement-mediated killing was assessed by incubating NTHi with serum and the role of the membrane-attack complex and classical/alternate pathways of complement activation measured. The effect of one strain to induce protective immunity against other strains was assessed. The effect of antibody on granulocyte intracellular killing of NTHi was also measured. The results showed that both healthy control subjects and bronchiectasis patients all had detectable antibody to NTHi of similar titre. Both groups demonstrated effective antibody/complement-mediated killing of different strains of NTHi. This killing was mediated through the membrane-attack complex and the classical pathway of complement activation. Immunization of rabbits with one strain of NTHi resulted in protection from other strains in vitro. Antibody activated granulocytes to kill intracellular bacteria. These findings may explain why NTHi rarely causes systemic disease in patients with chronic respiratory mucosal infection and emphasize the potential importance of cellular immunity against this bacterium.     

Experimental murine tularemia caused by Francisella tularensis, live vaccine strain: a model of acquired cellular resistance

We have established a model of experimentally-induced tularemia in mice, using the live vaccine strain of Francisella tularensis. A sublethal, intravenous inoculation of this organism caused in C57BL/6 strain mice an acute infection which lasted approximately 12 days. The clearance of Francisella from the bloodstream was shown to be complete by 5.5 hours postinfection. At this time, approximately twice as many bacteria were isolated from the spleen as from the liver. Mice which had recovered from a primary infection demonstrated a significant resistance to re-infection with autologous Francisella, a memory which persisted for at least 15 weeks. Resistance to experimental tularemia could be passively transferred from infected mice to naive mice by means of non-adherent spleen cells. Cells capable of adoptive transfer of resistance were present at a maximal concentration 7 days following infection, and persisted in significant numbers within the spleen cell population for at least 20 days after infection. Treatment of mice with serum from recovered animals caused a decrease in resistance when measured in the livers, and an increase in resistance when measured in the spleens. Suppression of T cell-mediated immunity during infection by treatment with cyclosporin A resulted in a dramatic increase in the tissue bacterial counts. Cyclosporin A-induced suppression of antitularemic resistance was first noted 2-3 days following infection and remained apparent for at least 8 days. The results of these experiments demonstrate that resistance to experimental murine tularemia is mediated predominantly by a cell-mediated mechanism. This mechanism involves T cells which become activated as early as 2-3 days following infection. Experimental, non-lethal infection with Francisella tularensis is thus an excellent model for investigating the mechanisms of acquired cellular immunity.