Handling of soluble IgA aggregates by the mononuclear phagocytic system in mice. A comparison with IgG aggregates.

We have studied the fate of soluble stable aggregates of human IgA (A-IgA) and IgG (A-IgG) after their injections in mice, as well as in vitro catabolism by liver, kidney and peritoneal macrophages. The half-life of the fast component of blood clearance was similar for both aggregates. However the half-life of the slow component of A-IgA clearance was significantly slower than A-IgG (t1/2 10 . 03 v. 7 . 52 hr, respectively). The A-IgA deposited in liver and kidney was removed significantly more slowly than A-IgG. Studies in isolated liver and kidney slices suggest that this could be due to the impaired catabolism of A-IgA as opposed to that of A-IgG. Interestingly the kidney hardly participates in the processing of A-IgA. At the three doses employed (1, 5 and 10 micrograms of both aggregates) peritoneal macrophages bound and catabolised significantly less amount of A-IgA than A-IgG. Complement seems to have no role in the processing of A-IgA by peritoneal macrophages unlike that observed with A-IgG. It is suggested that the impairment in handling of A-IgA by the mononuclear phagocytic system could provoke their persistence in the circulation and deposition at sites susceptible to injury. These results may be of some relevance for the understanding of physiological IgA-IC (immune complex) clearance and for the pathogenesis of IgA-related diseases.     

Blood clearance and tissue localization of soluble aggregates of IgG in NZB/W and NZB mice.

We studied the capacity of the mononuclear phagocytic system (MPS) of NZB/W and NZB mice to clear trace and saturating doses of soluble heat-aggregates of IgG (A-IgG) from the blood. Mature female NZB/W mice (aged 5-7 months) with early glomerulonephritis showed no differences in MPS clearance of A-IgG compared with younger NZB/W mice without glomerulonephritis. In contrast, mature NZB mice had a more rapid clearance of A-IgG and greater MPS localization of A-IgG than their younger counterparts. Further studies showed that older NZB/W mice (greater than 10 months) had a slightly more rapid clearance of A-IgG than 2-5-month-old mice (t 1/2 = 3.34 +/- 0.27 SEM vs 3.76 +/- 0.34 SEM, P less than 0.01), whereas NZB mice mice older than 10 months of age had a markedly more rapid clearance than 2-5-month-old NZB mice (t 1/2 = 2.84 +/- 0.15 SEM vs 3.76 +/- 0.32, P less than 0.005). The more rapid clearance seen in NZB mice was partly explained by greater splenic localization of A-IgG and appeared to be restricted to Fc- and/or C3b-receptor mediated clearance, in that clearance of aggregated albumin was not changed. We conclude that NZB/W mice have no impairment in MPS clearance capacity at the onset of their glomerulonephritis, and slightly increased clearance capacity late in the course of their disease. Thus, the presence of circulating immune complexes and the development of glomerulonephritis in NZB/W mice is unlikely to be due to a diminished MPS clearance capacity. NZB mice have an increase in MPS capacity to clear A-IgG as a function of age.     

Binding kinetics of monomeric and aggregated IgG to Kupffer cells and hepatocytes of mice

The binding kinetics of human monomeric IgG and stable heat-aggregated IgG (A-IgG) to Fc receptors of hepatocytes and Kupffer cells isolated from mice was studied. After injection of radiolabelled proteins the 60-70% of hepatic uptake was recovered in parenchymal cells (hepatocytes). In experiments in vitro the A-IgG bound in larger amounts to hepatocytes and Kupffer cells than monomeric IgG. The association rate constants of aggregates were somewhat higher for Kupffer cells than for hepatocytes whereas the percentage uptake of aggregates by Kupffer cells was only 5-15% of that of hepatocytes. The equilibrium constants of aggregates binding to both cells amounted to 0.4-1 X 10(8) M-1 for A-IgG compared with an equilibrium constant for monomeric IgG of 1-2 X 10(7)M-1. The maximum number of IgG and A-IgG molecules bound per cell was higher on hepatocytes (mean 14 X 10(6)) than on Kupffer cells (mean 2 X 10(5)) which is in agreement with the higher binding capacity of hepatocytes for these proteins observed in vivo and in vitro experiments. The ability to compete for receptor binding seemed to reside exclusively in the Fc portion of IgG since F(ab')2 fragments of IgG failed to inhibit labelled monomeric IgG or A-IgG. The receptor seems to be specific for IgG since unlabelled monomeric IgA demonstrated no binding inhibition of labelled IgG or A-IgG on hepatocytes and Kupffer cells. The overall results further suggest that hepatocytes might through Fc receptors play a collaborative role with the mononuclear phagocytic system in the clearance of circulating immune complexes.     

Cadmium transport in isolated perfused rat liver: zinc-cadmium competition.

The hypothesis that one component of cadmium uptake by rat hepatocytes involves a mediated transport pathway normally operative for zinc transport was tested in the isolated perfused rat liver preparation. Excess zinc in the perfusion medium suppressed cadmium uptake as indicated by the decrease in the normalized clearance (initial clearance divided by liver weight) from 0.340 +/- 0.019 (ml/min)/g in the presence of normal zinc concentrations (Zn:Cd molar ratio, 1.6) to 0.138 +/- 0.017 (ml/min)/g (Zn:Cd molar ratio, 13.0). In excess-zinc control experiments (no cadmium present) little zinc is accumulated by the liver, apparently due to competition between intrahepatic and extracellular binding. Exposure to cadmium increases both zinc secretion into the perfusion medium and biliary excretion of zinc. The effect at the sinusoidal membrane is probably a result of both the blockage of zinc resorption during cadmium uptake and the displacement of intrahepatic zinc. The effect on biliary excretion of zinc is due solely to displacement of intrahepatic zinc. These results are consistent with the proposed hypothesis for cadmium transport.     

Plasminogen activators direct reorganization of the liver lobule after acute injury

Tissue repair requires an adequate cellular proliferation coordinated with the timely proteolysis of matrix elements. Based on the properties of plasminogen activators in liver cell proliferation and tissue proteolysis, we explored the regulatory role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in liver repair. Using carbon tetrachloride (CCl(4)) intoxication as a model of acute liver injury, we found that tPA-deficient mice displayed a mild defect in hepatic repair, whereas livers of uPA-deficient mice had a more substantial delay in repair, with injury of centrilobular hepatocytes persisting up to 14 days after CCl(4). Notably, functional cooperativity between plasminogen activators was strongly inferred from the profound reparative defect in livers of mice lacking tPA and uPA simultaneously, with persistence of centrilobular injury as far out as 35 days. The defective repair was not because of increased susceptibility of experimental mice to the toxin or to inadequate cellular proliferation. Instead, lack of plasminogen activators led to the accumulation of fibrin and fibronectin within injured areas and poor removal of necrotic cells. These data demonstrate that tPA and uPA play a critical role in hepatic repair via proteolysis of matrix elements and clearance of cellular debris from the field of injury.     

The effect of the injection of a synthetic platelet activating factor (PAF-acether) on the fate of IgG aggregates in mice

The effect of the injection of a synthetic platelet-activating factor (PAF-acether) upon the fate of exogenous immune aggregates was studied in normal mice. When animals received 0.5 μg of PAF-acether following the administration of 5 μg heat-aggregated human IgG (A-IgG), a 50% increase of clearance velocity was observed during the initial 5 min; thereafter, between 15 and 30 min, there was a marked increase in blood levels of immune aggregates which paralleled the increase in plasma volume observed at those times. As regards the effect of the infusion of PAF-acether on the distribution of A-IgG in the organs, hepatic uptake was not affected, kidney uptake was reduced during the first 15 min, and lung and spleen showed only minor variations. When animals were injected with 0.5 μg PAF-acether, skin trapped aggregates showed a 73% increase above control values at 5 min, followed by a fall to control values coincidently with the flood back of the aggregates into the circulation and the increase in blood volume. The injection of lower doses of PAF-acether (0.05 μg) or Diazoxide (1.5 mg) did not induce any change on the skin trapping of aggregates. In conclusion, the powerful action of PAF-acether on vascular permeability allows a reversible deposition of immune aggregates in the skin which is followed by the flood back of this material into the circulation when its pharmacological action subsides. These findings may be of some relevance to the understanding of the dynamic processes involved in the deposition of immune complexes in tissues, especially as regards skin vessels.     

Aggregated human immunoglobulin G stabilized by albumin: a standard for immune complex detection.

Stabilized preparations of heat-aggregated human immunoglobulin G (A-IgG) of restricted size were made by separating A-IgG by sucrose density gradient ultracentrifugation in the presence of bovine serum albumin (BSA). Periodic recentrifugation of stored fractions of the radiolabelled A-IgG indicated that the initial sedimentation characteristics were preserved. Pooled fractions of A-IgG stored for up to 16 months had the same functional activity as freshly prepared A-IgG of corresponding size when assessed by activation of the first component of complement and consumption of C4 and CH50 in normal human serum. It was also found that the reactivity of A-IgG in the C1q binding assay (C1Q-BA) and the conglutinin binding assay (Con-BA) was not altered by long-term storage of these A-IgG. Testing different batches of [125I]C1q and conglutinin with the same batch of stabilized A-IgG showed variations due to the instability of both [125I]C1q and conglutinin. The influence of these variations on the quantification of the levels of immune complexes in sera was reduced by using stable A-IgG as a reference. The assays were compared to determine the effect of the size of the aggregate. The C1Q-BA detected preferentially A-IgG of large size, while size had no influence in the Con-BA. These results suggest that the stability of A-IgG in BSA is such that this preparation may be used as a reliable standard for immune complex assays.     

Effect of preformed immune complexes on the clearance and tissue localization of single-stranded DNA in mice.

Recent studies have shown that DNA is cleared from the circulation extremely rapidly by the liver, and that normal individuals have low or immeasurable levels of circulating DNA. In some patients with SLE and in NZB/W mice, however, significant amounts of free DNA as well as DNA-anti-DNA immune complexes have been found in the circulation, suggesting a possible defect in DNA clearance in these conditions. To delineate factors which might contribute to the persistence of DNA in the circulation, we have assessed the effects of immune complexes on the clearance of single stranded DNA in normal C57Bl/6J mice. HSA-anti-HSA immune complexes at five-fold antigen excess were injected intravenously and after a variable, the clearance of single-stranded DNA was determined. Clearance of all doses of DNA was markedly suppressed 6 to 12 hr after the administration of immune complexes and returned to normal by 24 hr. Immune complexes decreased DNA clearance by blocking the hepatic uptake of DNA without altering the distribution of DNA to other organs. Histology and studies on the effect of immune complexes on the clearance of bromosulphophthalein (BSP) and sulphur colloid suggest that immune complexes affect DNA clearance by altering hepatic blood flow. The results obtained in this study suggest that circulating immune complexes in patients with SLE or in other conditions may suppress normal DNA clearance, and thereby contribute to the persistence of DNA in the circulation.     

Improved standardization in the quantitative estimation of soluble immune complexes making use of an international reference preparation. Results of a collaborative multicentre study.

Heat aggregated immunoglobulin G (A-IgG) of restricted size and complement solubilized tetanus toxoid (Te): human anti-Te immune complexes (IC) were sent as coded test samples to eight laboratories for quantitative assessment by different IC assay techniques including C1q solid and fluid phase binding assays, conglutinin binding assay. Raji cell test and particle counting immunoassay. In addition, samples containing the same material at concentrations communicated to the laboratories for the performance of reference curves were included. The investigators were asked to estimate the quantity of A-IgG or Te:aTe in the coded samples by reference to both their own locally produced standards and to the A-IgG and Te:aTe reference preparations of known concentrations. When calculated on the basis of locally prepared standards the range of concentrations found by the various laboratories and tests was 20-260 micrograms/ml for A-IgG (actual concentration 50 micrograms/ml) and 32-1,420 micrograms/ml for Te:aTe complexes (actual concentration 40 micrograms complexed antibody/ml). When read on the international candidate reference A-IgG preparation these ranges were 28-800 micrograms/ml and 35-800 micrograms/ml, respectively. The highest standardization efficiency was obtained when the Te:aTe reference curve was used for quantitation: the range of results obtained for A-IgG and Te:aTe coded samples being as narrow as 7-40 micrograms/ml and 34-68 micrograms/ml, respectively. Thus, when the content of the coded samples was estimated on the basis of the Te:aTe reference curves established in the laboratories a narrow clustering of the results was seen. It is proposed that the Te:aTe preparation, which has been found stable during storage for 2 years, could serve as a useful international reference preparation in the field of IC determination.     

Defective hepatic handling of IgA immune aggregates by mice with experimental IgA nephropathy.

In an experimental model of IgA nephropathy induced in mice by chronic immunization with dextran, we tested the hypothesis that a defect in the hepatic handling of IgA could be an important determinant in the deposition of IgA in the mesangium. In mice injected with 1-16 doses of 1 mg of dextran (after a preimmunization period of 21 days) the blood clearance of IgA immune aggregates was significantly delayed in relation to control animals, becoming normal at 24 injections. This alteration seems specific since the clearance of IgG aggregates was normal. The percentage of isolated hepatocytes with Fc receptors for IgA decreased significantly over the whole period of dextran immunization. The binding rate of 125I-IgA aggregates to hepatocytes of mice with 24 dextran injections was twice lower than that of control animals. By contrast, the percentage of Kupffer cells with IgA receptors increased over ensuing dextran injections. A progressive increase in the IgA blood levels and in the percentage of mice with mesangial IgA deposits was seen along the period of study. At 24 injections most animals presented moderate to intense mesangial proliferation and abundant electron-dense deposits. On the whole, these data suggest that the early impairment in the liver IgA clearance capacity observed in these animals could facilitate the presence of circulating immune complexes (IC) and their deposition in the mesangium. The increase in serum IgA, seen thereafter, together with the normalization of the IgA clearance capacity, suggest that other pathophysiological mechanism(s) (e.g. in situ IC formation or IgA polymers deposition) must also be involved in this model of experimental IgA nephropathy.     

Defects in mononuclear phagocytic system (MPS) function in autoimmune MRL-lpr/lpr mice

MRL-lpr/lpr mice develop an autoimmune disease similar to systemic lupus erythematosus. To determine whether mice of this strain develop defects in mononuclear phagocyte system (MPS) function similar to those observed in patients, the pattern of sequestration of labeled immune complexes was compared 90 min after infusion into MRL-lpr/lpr and into normal B6D2 mice. The amount of complexes persisting in the blood was increased, and the amount sequestered in the liver was significantly reduced in MRL-lpr/lpr mice in comparison to normal B6D2 controls. This defect was most evident in MRL-lpr/lpr mice of the ages of 25-26 weeks; mice of this age also demonstrated the greatest elevation of anti-DNA antibody levels. The role of the MRL strain background and of the lpr gene in determining this defect was investigated by analysis of MRL-+/-/+/- and of other lpr congenic strains (B6-lpr/lpr, AKR-lpr/lpr, and C3H-lpr/lpr). Both MRL-+/-/+/- and congenic lpr animals showed similar defects, although to a lesser degree than MRL-lpr/lpr mice. In contrast, MRL-lpr/lpr mice demonstrated normal clearance of heat-damaged red blood cells and heat-aggregated albumin. Thus MRL-lpr/lpr mice display a selective defect in MPS Fc receptor function and may provide a valuable model for elucidating the etiology and importance of MPS dysfunction in immune complex deposition disease.     

Chromatin clearance in C57Bl/10 mice: interaction with heparan sulphate proteoglycans and receptors on Kupffer cells.

Chromatin is an important autoantigen in the pathogenesis of systemic lupus erythematosus (SLE) as an immunogen and as a part of nephritogenic immune complexes. Earlier studies focused on clearance of DNA. However, DNA released into the circulation from dying cells is found associated with histones in nucleosomes. The liver is the major organ involved in clearance of chromatin from the circulation of mice. Heparan sulphate proteoglycans (HSPG) have been implicated in the clearance of various charged molecules. Receptor-mediated clearance of ssDNA by the liver has also been reported. Because chromatin contains positively charged histones in addition to DNA, we wished to determine if HSPG and/or DNA receptors are involved in chromatin clearance. The rate of clearance of H1-stripped chromatin from the bloodstream of C57Bl/10 mice was markedly decreased by prior treatment of mice with Heparinase I. Clearance was also inhibited by heparin, heparan sulphate, and DNA, but not by colominic acid. DNA was the most effective inhibitor of clearance and released chromatin from sites of clearance. Depletion of Kupffer cells and splenic macrophages using liposome-encapsulated Clodronate (dichloromethylene bisphosphonate) markedly inhibited chromatin clearance. These data suggest that chromatin clearance is mediated by charge interactions with cell surface HSPG and by DNA receptors. Clearance and degradation of chromatin require functional macrophages in the liver and spleen.     

A deficiency in the in vivo clearance of apoptotic cells is a feature of the NOD mouse

Deficiencies in apoptotic cell clearance have been linked to autoimmunity. Here we examined the time-course of peritoneal macrophage phagocytosis of dying cells following the direct injection of apoptotic thymocytes into the peritoneum of NOD mice and BALB/c controls. Macrophages from NOD mice demonstrated a profound defect in the phagocytosis of apoptotic thymocytes as compared to control macrophages. Nonobese diabetic mice also demonstrated a decrease in the clearance of apoptotic cell loads following an apoptotic stimulus to thymocytes (dexamethasone) when compared to BALB/c or NOR controls. Further, NOD mice demonstrated an increase in apoptotic cell load following an apoptotic stimulus to keratinocytes (ultraviolet light, UVB) when compared to control strains. Animals deficient in macrophage phagocytosis of apoptotic debris often manifest an autoimmune phenotype characterized by the production of antinuclear autoantibodies (ANA). We determined whether increased apoptotic cell loads (through repeated exposure to UVB irradiation) could accelerate such autoimmune phenomena in young NOD mice. Following repeated UVB irradiation, NOD mice, but not BALB/c or NOR controls, developed ANA. We propose that abnormalities in apoptotic cell clearance by macrophages predispose NOD mice to autoimmunity.     

Clearance of sensitized erythrocytes in NZB/NZW mice Effects of castration and sex hormone treatment

The clearance of particulate immune complexes consisting of erythrocytes sensitized with IgG or complement was investigated in (NZB × NZW)F₁ (B/W) mice. Treatment of castrated B/W mice with androgen or estrogen was able to modulate this clearance. Young (3-month-old) male and female B/W mice cleared IgG-sensitized mouse erythrocytes rapidly, whereas older males (13 months) and females (7 months) showed a marked impairment in their ability to clear these cells. In addition, erythrocytes sensitized with complement in the absence of antibody were cleared within 5 min in young B/W mice. Older mice showed a greater and more rapid clearance rate of these cells. Castrated female B/W mice treated with androgen implants from three weeks of age showed improved clearance of IgG-sensitized erythrocytes at 7 months, whereas estrogen-treated male mice showed delayed clearance. These results suggest an age-dependent defect in the clearance of IgG-sensitized particles, perhaps due to diminished levels of serum complement and/or saturation of Fc receptors. In addition, there is an alteration in the clearance of complement-sensitized erythrocytes which may be related to changes in macrophage activity or enzyme inactivators of C3 and C4. The possible mechanisms responsible for the hormonal modulation of clearance are discussed in relation to the known ability of these hormones to influence autoimmune diseases.     

Characteristics of macrophages in irradiation chimeras in mice reconstituted with allogeneic bone marrow cells

Biological and immunological characteristics of the reticuloendothelial system of irradiation bone marrow chimeric mice and macrophages collected from various tissue sources of the mice were studied. The chimeras showed comparable activities in carbon clearance to those of normal donor or recipient mice. The macrophages from spleen, lymph node, bone marrow, peripheral blood, liver, peritoneal cavity, and lung were demonstrated to be of donor marrow origin. They showed almost the same enzyme activities and phagocytic capability of sheep erythrocytes (SRBC, E), SRBC sensitized with anti-SRBC IgG (EA), and SRBC sensitized with anti-SRBC IgM and coated with complement (EAC) as those of normal mice. Proportions of Fc receptor and complement receptor-positive cells are also in normal range. In addition, the antigen-presenting capability of the chimeric macrophages for in vitro primary antibody response to SRBC was intact. These observations suggest that the reticuloendothelial system and macrophages of allogeneic bone marrow chimeras where donor and recipient differ at the major histocompatibility complex have no defect so far as could be ascertained by the present study.     

Association of Heme Oxygenase 1 with the Restoration of Liver Function after Damage in Murine Malaria by Plasmodium yoelii

The liver efficiently restores function after damage induced during malarial infection once the parasites are cleared from the blood. However, the molecular events leading to the restoration of liver function after malaria are still obscure. To study this, we developed a suitable model wherein mice infected with Plasmodium yoelii (45% parasitemia) were treated with the antimalarial α/β-arteether to clear parasites from the blood and, subsequently, restoration of liver function was monitored. Liver function tests clearly indicated that complete recovery of liver function occurred after 25 days of parasite clearance. Analyses of proinflammatory gene expression and neutrophil infiltration further indicated that hepatic inflammation, which was induced immediately after parasite clearance from the blood, was gradually reduced. Moreover, the inflammation in the liver after parasite clearance was found to be correlated positively with oxidative stress and hepatocyte apoptosis. We investigated the role of heme oxygenase 1 (HO-1) in the restoration of liver function after malaria because HO-1 normally renders protection against inflammation, oxidative stress, and apoptosis under various pathological conditions. The expression and activity of HO-1 were found to be increased significantly after parasite clearance. We even found that chemical silencing of HO-1 by use of zinc protoporphyrin enhanced inflammation, oxidative stress, hepatocyte apoptosis, and liver injury. In contrast, stimulation of HO-1 by cobalt protoporphyrin alleviated liver inflammation and reduced oxidative stress, hepatocyte apoptosis, and associated tissue injury. Therefore, we propose that selective induction of HO-1 in the liver would be beneficial for the restoration of liver function after parasite clearance.     

The effect of humoral and cell-mediated immunity in resistance to systemic Serratia infection.

Protection against experimental Serratia marcescens infection in mice was enhanced by prior injection of formalin-killed or viable bacteria of the same strain. From the first to the fourth week after vaccination, specific immunity was involved in the host defence against systemic serratia infection. The transfer of antiserum specific for S. marcescens increased bacterial clearance from the liver, but did not increase the survival of the mice. Bacterial clearance from the liver was also increased by the transfer of spleen cells from immunised mice, but, again, survival was not increased. However, the transfer of both antiserum and spleen cells from vaccinated mice increased both bacterial clearance from the liver and survival (p less than 0.01). These results suggest an additive effect of humoral immunity and T-cell-mediated immunity in protection against systemic serratia infection.     

Effect of cyclophosphamide on the clearance of IgG-sensitized red cells in mice

The effects of cyclophosphamide (Cy) on the clearance of IgG-sensitized erythrocytes (EA) were examined. The results clearly indicate that Cy treatment enhances the capacity of the mononuclear phagocytic system to remove antibody-coated cells from the circulation in normal and decomplemented mice. The enhanced rate of clearance is the consequence of an increased uptake of EA by the liver and spleen. We explored the possibility that the enhancement of Fc receptor-mediated clearance might be an important effect to be taken into account in the search for a more effective therapy of immune complexes diseases.     

Isolation of Model Soluble Immune Complexes in the Fluid Phase by Monoclonal Anti-IgG- Ferritin Antibodies

Ferritin conjugates of monoclonal IgG anti-human gamma chain (anti-IgG-F) were reacted with soluble heat aggregates of IgG (A-IgG) and with soluble DNA-anti-DNA complexes to increase the S rate of the model soluble immune complexes (ICx) and thus facilitate isolation of ICx in the fluid phase and provide an immunochemical marker for subsequent ultrastructural analysis. A-IgG appeared as globular or curvilinear structures with individual IgG molecules arranged in a random fashion. The technique appears promising for characterization of other soluble ICx.     

Defective clearance of adenovirus in IRF-1 mice associated with defects in NK and T cells but not macrophages

A replication-defective adenovirus-LacZ recombinant virus (AdLacZ) was injected intravenously into IRF-1(-/-) mice and wild-type mice to characterize the contribution of IRF-1 to the immune-mediated clearance of Ad vector. Compared with wild-type mice, IRF-1(-/-) mice expressed higher levels of the LacZ gene product in the liver. After infusion of the AdLacZ, the expression of IRF-1 mRNA was upregulated in the liver of wild-type mice, but not in IRF-1(-/-) mice. Both spleen and liver mononuclear cells from IRF-1(-/-) mice initially exhibited a markedly lower number of NK, NK-T and CD8 T cells. At day 7 after the administration of AdLacZ, there was a significantly increased population of NK, NK-T and CD8 T cells in both spleen and liver, and also CD11b(+) cells in liver of IRF-1(-/-) mice, compared with the increased in wild-type mice. As IRF-1 is an important signal for production of IFN-gamma by CD8 T and NK cells as well as production of IL-12 by CD11b(+) cells, we determined whether there were lower levels of these cytokines in IRF-1(-/-) mice after Ad challenge. Surprisingly, there were lower levels of IL-12, but higher levels of IFN-gamma and IL-18 in IRF-1(-/-) compared with wild-type mice at day 7 after administration with AdLacZ. These results indicate that delayed clearance of Ad is associated with partial correction of defects of the NK, NK-T and CD8 T cells and increased production of IFN-gamma and IL-18 in IRF-1(-/-) mice.