Suppressor function of hepatic mononuclear inflammatory cells during murine chronic graft-vs-host disease. I. Macrophage-enriched cells mediate suppression in the liver.

Murine chronic graft-vs-host disease (CGBHD) to minor histocompatibility antigens (B10.D2----BALB/c) is characterized by inflammatory destruction of intrahepatic bile ducts, scleroderma-like skin lesions, and lymphoid involution. Spleen cells isolated from this model proliferate poorly when stimulated with mitogens. Previous reports indicate defective lymphocyte proliferation in this model is the result of active suppression induced by the graft-vs-host reaction in the spleen and is mediated by Thy 1.2-, sIg-, plastic nonadherent, splenic natural suppressor (NS) cells. To determine whether the intense CGVHD in the liver is associated with induction of suppression, we compared the suppressor activity of hepatic and splenic mononuclear inflammatory cells isolated concurrently during murine CGVHD. Both hepatic and splenic MC suppressed the proliferation of mitogen-stimulated normal spleen cells in a non-MHC, non-Mls restricted manner. T cells contributed to the suppressor activity of both populations. However, the suppressor activity of hepatic MC was mediated largely by a macrophage-enriched population of MC while that of splenic MC was mediated largely by NS cells.     

Suppressor function of liver mononuclear cells isolated during murine chronic graft-vs-host disease. II. Role of prostaglandins and interferon-gamma.

Mononuclear inflammatory cells (MC) isolated from the livers and spleens of mice with chronic graft-vs-host disease (CGVHD) to minor histocompatibility antigens (B10.D2----BALB/c) show defective proliferation when stimulated with Con A and LPS. In turn, both CGVHD liver and spleen cells suppress the proliferation of mitogen-stimulated normal spleen cells in a genetically unrestricted manner. The suppressor activity of CGVHD spleen cells is mediated by plastic nonadherent null (natural suppressor) cells and involves a soluble suppressor factor(s). In contrast, the suppressor activity of CGVHD liver cells is mediated by macrophages (M phi). In the current studies we show that the suppressor activity of CGVHD liver cells is also mediated by soluble factors and compare the roles of prostaglandins and interferon (IFN)-gamma in mediating defective proliferation and suppressor activities of CGVHD liver and spleen MC. Monoclonal antibody to IFN-gamma partially reversed the defective mitogen-stimulated proliferation of CGVHD spleen MC but had no effect on proliferative response of CGVHD liver MC. Indomethacin did not alter the low proliferative response of either CGVHD liver or spleen MC. Anti-IFN-gamma inhibited the ability of CGVHD spleen cells to suppress proliferation of Con A and LPS-stimulated B10.D2 spleen cells. In contrast, anti-IFN-gamma resulted in a small decrease in the ability of liver MC to suppress Con A (but not LPS)-stimulated cell proliferation. Indomethacin decreased the ability of both CGVHD liver and spleen cells to suppress Con A-stimulated proliferation but had inconsistent effects on LPS-stimulated proliferation. These results show that IFN-gamma and prostaglandins partially mediate the suppressor activity of CGVHD spleen MC. The suppressor activity of CGVHD liver MC also involves prostaglandins but is relatively independent of IFN-gamma.     

Immune dysfunction associated with graft-vs-host reaction in mice transplanted across minor histocompatibility barriers. II. Reversible defect in T-dependent antibody responses

B cell and Th cell functions were assessed in mice undergoing a graft-vs-host reaction (GvHR) in response to minor histocompatibility Ag by using the plaque-forming cell (PFC) response to the T-independent Ag TNP-Brucella abortus and the T-dependent Ag TNP-SRBC. Bone marrow plus spleen cells from B10.D2 mice were transplanted into lethally irradiated B10.D2 (syngeneic recipient) or H-2d-compatible BALB/c (allogeneic recipient) to produce a chronic form of GvHR. BALB/c recipients of an allogeneic transplant demonstrated a marked and proportional lymphoid depletion of the spleen with normal percentages of B cells, T cells, and CD4+ and CD8+ T cell subsets. Mice with GvHR made normal numbers of PFC/10(5) spleen cells in response to the T-independent Ag, but a significantly depressed number of PFC/10(5) spleen cells to the T-dependent Ag compared with normal B10.D2 mice and with irradiated B10.D2 recipients of syngeneic B10.D2 marrow plus spleen cells. Mice undergoing the minor Ag GvHR made significantly larger numbers of PFC/10(5) spleen cells after secondary immunization with TNP-SRBC compared with controls. In vitro assays demonstrated that B cells from mice with GvHR responded to T help from normal B10.D2 mice and that T cells from mice with GvHR provided help to normal B cells after in vivo immunization. These data demonstrate that radiation chimeras with GvHR in response to minor histocompatibility Ag have relatively normal B cell function and an apparent defect in T helper cell function that is reversible by immunization with appropriate Ag.     

哺乳期BALB/c小鼠胆道梗阻模型的探索

目的探讨新生BALB/c小鼠胆道梗阻模型的建立,并与报告的新生BALB/c小鼠感染猕猴轮状病毒(RRV)模型小鼠生存曲线进行比较。方法将出生后5～7 d的BALB/c小鼠随机分为实验组和对照组,实验组进行胆总管结扎,然后关腹。对照组打开腹部后关腹不结扎胆总管。实验完成后每天观察小鼠的体重变化、无毛区皮肤颜色变化、小鼠存活天数以及在术后第5、10天时分别取小鼠肝脏做病理及免疫组化。结果小鼠在结扎后随着时间的延长,小鼠的体重及肝体比、无毛区皮肤颜色、存活天数、肝脏病理等都存在一定变化。小鼠体重增长逐渐缓慢,术后第2天就会出现无毛区的皮肤变黄,在尿道口会有淡黄色的液体并随后出现陶土样便。在术后第5天及第10天时取肝脏做肝体比有统计学差异(P≤0.05),小鼠在术后第10天左右会出现死亡高峰。结论新生BALB/c小鼠胆总管结扎模型是研究胆道梗阻的可靠动物实验,其生存曲线与报告的猕猴轮状病毒致胆道闭锁大体类似。     

Interaction of CD44 and hyaluronic acid enhances biliary epithelial proliferation in cholestatic livers

Biliary epithelia express high levels of CD44 in hepatobiliary diseases. The role of CD44-hyaluronic acid interaction in biliary pathology, however, is unclear. A rat model of hepatic cholestasis induced by bile duct ligation was employed for characterization of hepatic CD44 expression and extracellular hyaluronan distribution. Cell culture experiments were employed to determine whether hyaluronan can regulate cholangiocyte growth through interacting with adhesion molecule CD44. Biliary epithelial cells were found to express the highest level of CD44 mRNA among four major types of nonparenchymal liver cells, including Kupffer, hepatic stellate, and liver sinusoidal endothelial cells isolated from cholestatic livers. CD44-positive biliary epithelia lining the intrahepatic bile ducts were geographically associated with extracellular hyaluronan accumulated in the portal tracts of the livers, suggesting a role for CD44 and hyaluronan in the development of biliary proliferation. Cellular proliferation assays demonstrated that cholangiocyte propagation was accelerated by hyaluronan treatment and antagonized by small interfering RNA CD44 or anti-CD44 antibody. The study provides compelling evidence to suggest that proliferative biliary epithelia lining the intrahepatic bile ducts are a prime source of hepatic CD44. CD44-hyaluronan interaction, by enhancing biliary proliferation, may play a pathogenic role in the development of cholestatic liver diseases.     

Mouse strain difference in bile duct lesions induced by swine serum injections

The mouse strain difference in bile duct lesions was studied on male A/J, BALB/c, C57BL/6, C3H/He, DBA/2 and DDY mice 4 weeks old given intraperitoneal injections of swine serum (0.05 or 0.2 ml per mouse) twice a week for 4 weeks. The hepatic lesions were restricted to the portal tract. Biliary epithelial cells showed hypertrophy and hyperplasia, and eosinophilic and homogeneous or needle-shaped material appeared in the cytoplasm of such hypertrophied epithelial cells and in the ductular lumen. Around these damaged biliary epithelia, eosinophil leukocyte and plasma cell infiltration with proliferation of collagen fibres was commonly detected. These changes became more apparent with increasing size of bile duct. Such histopathological characteristics of hepatic lesions were essentially the same in all strains, but the severity showed a clear strain difference: the lesion was marked in the DDY, A/J and BALB/c strains, moderate in C3H/He and slight in C57BL/6 and DBA/2. A high production of anti-swine-serum antibodies associated with a marked increase in the number of mouse IgG-producing lymphocytes in the spleen was detected in the strains showing the marked hepatic lesions.     

Drug-induced bile duct injury

Drug-induced liver injury includes a spectrum of pathologies, some related to the mode of injury, some to the cell type primarily damaged. Among these, drug-induced bile duct injury is characterized by the destruction of the biliary epithelium following exposure to a drug. Most of the drugs associated with bile duct injury cause immune-mediated lesions to the epithelium of interlobular ducts. These share common histopathological features with primary biliary cholangitis, such as inflammation and necrosis at the expense of cholangiocytes and, if the insult persists, bile duct loss and biliary cirrhosis. Some drugs selectively target larger ducts. Such injury is often dose-dependent and thought to be the result of intrinsic drug toxicity. The histological changes resemble those seen in primary sclerosing cholangitis. This overview focuses on the clinical and pathological features of bile duct injury associated with drug treatment and on the immunological and biochemical effects that drugs exert on the biliary epithelium. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

The existence of antibody-like activities against the weakly immunogenic minor histocompatibility antigens

BALB/c mice develop cytotoxic lymphocytes as well as produce specific antibodies against the minor histocompatibility antigens when injected with DBA/2 P815 cells. P815 cells grown in BALB/c mice have IgG antibodies on their surface as demonstrated by the binding of ¹²⁵I-labeled goat anti-mouse IgG and by complement-dependent cytotoxicity. Serum from BALB/c mice hyperimmunized with P815 cells blocked lymphocyte-mediated cytotoxicity by BALB/c immune peritoneal exudate cells. This blocking activity was removed by absorbing hyperimmune serum with DBA/2 spleen cells or P815 cells. This result suggests that specific antibodies were generated against the minor histocompatibility differences between BALB/c and DBA/2 mice. The experimental procedures described may be very useful in demonstrating minute quantities of antibody against minor histocompatibility antigens on tumor cells.     

Immunohistochemical and immunoelectron microscopic analyses of alpha-amylase isozymes in human intrahepatic biliary epithelium and hepatocytes.

The expression and localization of the pancreatic and salivary isozymes of alpha-amylase in the intrahepatic biliary epithelium and hepatocytes were examined by the immunohistochemical method with polyclonal and monoclonal antibodies in 45 normal autopsied human livers. Immunoelectron microscopic studies with the protein A-gold method were performed with the monoclonal antibodies (MAb) on seven of the livers. The intrahepatic biliary system was divided into large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands. Immunohistochemically, pancreatic isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands in almost all livers. Interlobular ducts expressed pancreatic isozyme in only four (9%) livers. Bile ductules and hepatocytes were negative for pancreatic isozyme in all cases. Expression of salivary isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands in almost all livers, although the expression in interlobular ducts and bile ductules was weak. Hepatocytes were weakly positive for salivary isozyme. Immunoelectron microscopy revealed that both pancreatic and salivary isozymes were located in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands, and that hepatocytes had no pancreatic isozyme but contained salivary isozyme. These data suggest that pancreatic and salivary isozymes of alpha-amylase are produced by the intrahepatic biliary epithelium and secreted into intrahepatic biliary lumens, and that they may play an important role in the physiology of the intrahepatic biliary tree and hepatic bile. It is also suggested that hepatocytes produce a small amount of salivary alpha-amylase that may be secreted into the biliary tree.     

Protein composition of liver cyst fluid from the BALB/c‐cpk/+ mouse model of autosomal recessive polycystic kidney disease

Cysts arising from hepatic bile ducts are a common extra‐renal pathology associated with polycystic kidney disease in humans. As an initial step in identifying active components that could contribute to disease progression, we have investigated the protein composition of hepatic cyst fluid in an orthologous animal model of autosomal recessive polycystic kidney disease, heterozygous (BALB/c‐cpk/+) mice. Proteomic analysis of cyst fluid tryptic digests using LC‐MS/MS identified 303 proteins, many of which are consistent with enhanced inflammatory cell processes, cellular proliferation, and basal laminar fibrosis associated with the development of hepatic bile duct cysts. Protein identifications have been submitted to the PRIDE database ( http://www.ebi.ac.uk/pride ), accession number 9227.     

Biliary obstruction selectively expands and activates liver myeloid dendritic cells

Obstructive jaundice is associated with immunologic derangements and hepatic inflammation and fibrosis. Because dendritic cells (DCs) play a major role in immune regulation, we hypothesized that the immunosuppression associated with jaundice may result from the functional impairment of liver DCs. We found that bile duct ligation (BDL) in mice expanded the myeloid subtype of liver DCs from 20 to 80% of total DCs and increased their absolute number by >15-fold. Liver myeloid DCs following BDL, but not sham laparotomy, had increased Ag uptake in vivo, high IL-6 secretion in response to LPS, and enhanced ability to activate T cells. The effects of BDL were specific to liver DCs, as spleen DCs were not affected. Expansion of liver myeloid DCs depended on Gr-1(+) cells, and we implicated monocyte chemotactic protein-1 as a potential mediator. Thus, obstructive jaundice selectively expands liver myeloid DCs that are highly functional and unlikely to be involved with impaired host immune responses.     

Expression of neural cell adhesion molecule in human liver development and in congenital and acquired liver diseases

In the liver, neural cell adhesion molecule (NCAM) is a marker of immature cells committed to the biliary lineage and is expressed by reactive bile ductules in human liver diseases. We investigated the possible role of NCAM in the development of intrahepatic bile ducts and aimed at determining whether immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases. Therefore, we performed immunohistochemistry for NCAM and bile duct cell markers cytokeratin 7 and cytokeratin 19 on frozen sections of 85 liver specimens taken from 14 fetuses, 10 donor livers, 18 patients with congenital liver diseases characterized by ductal plate malformations (DPMs), and 43 cirrhotic explant livers. Duplicated ductal plates and incorporating bile ducts during development showed a patchy immunoreactivity for NCAM, while DPMs were continuously positive for NCAM. Bile ducts showing complete or patchy immunoreactivity for NCAM were found in cirrhotic livers, with higher frequency in biliary than in posthepatitic cirrhosis. Our results suggest that NCAM may have a function in the development of the intrahepatic bile ducts and that NCAM-positive immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases.     

LPS signaling enhances hepatic fibrogenesis caused by experimental cholestasis in mice

Although it is clear that bile acid accumulation is the major initiator of fibrosis caused by cholestatic liver disease, endotoxemia is a common side effect. However, the depletion of hepatic macrophages with gadolinium chloride blunts hepatic fibrosis. Because endotoxin is a key activator of hepatic macrophages, this study was designed to test the hypothesis that LPS signaling through CD14 contributes to hepatic fibrosis caused by experimental cholestasis. Wild-type mice and CD14 knockout mice (CD14(-/-)) underwent sham operation or bile duct ligation and were killed 3 wk later. Measures of liver injury, such as focal necrosis, biliary cell proliferation, and inflammatory cell influx, were not significantly different among the strains 3 wk after bile duct ligation. Markers of liver fibrosis such as Sirius red staining, liver hydroxyproline, and alpha-smooth muscle actin expression were blunted in CD14(-/-) mice compared with wild-type mice after bile duct ligation. Despite no difference in lymphocyte infiltration, the macrophage/monocyte activation marker OX42 (CD11b) and the oxidative stress/lipid peroxidation marker 4-hydroxynonenal were significantly upregulated in wild-type mice after bile duct ligation but not in CD14(-/-) mice. Increased profibrogenic cytokine mRNA expression in the liver after bile duct ligation was significantly blunted in CD14(-/-) mice compared with the wild type. The hypothesis that LPS was involved in experimental cholestatic liver fibrosis was tested using mice deficient in LPS-binding protein (LBP(-/-)). LBP(-/-) mice had less liver injury and fibrosis (Siruis red staining and hydroxyproline content) compared with wild-type mice after bile duct ligation. In conclusion, these data demonstrate that endotoxin in a CD14-dependent manner exacerbates hepatic fibrogenesis and macrophage activation to produce oxidants and cytokines after bile duct ligation.     

Hymenolepis microstoma: Correlation of histopathological host response and organ hypertrophy

The histological host response of female CF-1 mice to Hymenolepis microstoma involves dramatic changes in the host's bile duct and liver. These changes are similar to those described by previous investigators using different strains of mice. The histological changes which occur in bile duct and liver tissues of female CF-1 mice are accompanied by a significant hypertrophy of these organs; the wet weights of the bile duct and liver increase almost 2000 and 50%, respectively, in infected mice. No significant histopathological changes are found in the spleen or small intestine tissues of infected mice, yet the wet weight of both of these organs increases almost 100%. The magnitude of the histopathological changes which occur in the bile duct could possibly account for the increase in wet weight, i.e., there is significant fibrosis, but the histological changes in the liver, spleen, and small intestine do not appear to be of sufficient magnitude to account for the increases in wet weight.     

The homeobox gene Hhex is essential for proper hepatoblast differentiation and bile duct morphogenesis

Hhex is required for early development of the liver. A null mutation of Hhex results in a failure to form the liver bud and embryonic lethality. Therefore, Hhex null mice are not informative as to whether this gene is required during later stages of hepatobiliary morphogenesis. To address this question, we derived Hhex conditional null mice using the Cre-loxP system and two different Cre transgenics (Foxa3-Cre and Alfp-Cre). Deletion of Hhex in the hepatic diverticulum (Foxa3-Cre;Hhex(d2,3/-)) led to embryonic lethality and resulted in a small and cystic liver with loss of Hnf4alpha and Hnf6 expression in early hepatoblasts. In addition, the gall bladder was absent and the extrahepatic bile duct could not be identified. Loss of Hhex in the embryonic liver (Alfp-Cre;Hhex(d2,3/-)) caused irregular development of intrahepatic bile ducts and an absence of Hnf1beta in many (cystic) biliary epithelial cells, which resulted in a slow, progressive form of polycystic liver disease in adult mice. Thus, we have shown that Hhex is required during multiple stages of hepatobiliary development. The altered expression of Hnf4alpha, Hnf6 and Hnf1beta in Hhex conditional null mice suggests that Hhex is an essential component of the genetic networks regulating hepatoblast differentiation and intrahepatic bile duct morphogenesis.     

Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta

The inactivation of the Hnf1beta gene identified an essential role in epithelial differentiation of the visceral endoderm and resulted in early embryonic death. In the present study, we have specifically inactivated this gene in hepatocytes and bile duct cells using the Cre/loxP system. Mutant animals exhibited severe jaundice caused by abnormalities of the gallbladder and intrahepatic bile ducts (IHBD). The paucity of small IHBD was linked to a failure in the organization of duct structures during liver organogenesis, suggesting an essential function of Hnf1b in bile duct morphogenesis. Mutant mice also lacked interlobular arteries. As HNF1beta is not expressed in these cells, it further emphasizes the link between arterial and biliary formation. Hepatocyte metabolism was also affected and we identified hepatocyte-specific HNF1beta target genes involved in bile acids sensing and in fatty acid oxidation.     

Sphingosine-1-phosphate receptor agonists suppress concanavalin A-induced hepatic injury in mice

T cell-mediated immune responses play a critical role in a variety of liver injuries including autoimmune hepatitis. Injection of concanavalin A (Con A) into mice mimics the histological and pathological phenotype of T cell-mediated hepatitis. Recent advances in host immune control of organ transplantation include the development of sphingosine-1-phosphate (S1P) receptor agonists such as FTY720, which alter lymphocyte homing but do not suppress host general immunity. Herein we examined the effect of the new S1P receptor agonist KRP-203 on the Con A-induced liver damage model. In normal liver lymphocytes of BALB/c mice, both FTY720 and KRP203 promoted lymphocyte sequestering from the liver to secondary lymph nodes and significantly reduced the number of liver lymphocytes (p<0.05). Based on this observation, KRP203 was employed in the Con A-induced hepatitis model. KRP203 markedly reduced the number of CD4(+) lymphocytes that infiltrate Con A-treated liver (p<0.05) and successfully reduced serum transaminase elevation (p=0.017), therefore protecting mice from Con A-induced liver injury. Interestingly this homing modulation less occurs in natural hepatic T cell homing through the chemokine receptor, CXCR4. Therefore, S1P receptor agonists preferentially target CXCR4(+)CD4(+) peripheral blood T lymphocytes and suppress the occurrence of Con A-induced hepatitis, suggesting their therapeutic usefulness against T cell-mediated hepatic injury.     

Effect of graft-versus-host disease on anti-tumor immunity

BCL1, a spontaneous B cell leukemia of BALB/c origin, is rejected by C.B-20 (Ighb, H-40b) but not BALB/c (Igha, H-40a) mice. Adoptive transfer of C.B-20 anti-BCL1 effector cells specific for the minor histocompatibility Ag H-40a protects irradiated C.B-20 but not BALB/c recipients. Because C.B-20 donor cells could potentially generate graft-vs-host disease (GVHD) in BALB/c recipients, we investigated the possibility that GVHD prevents the anti-tumor effect. GVHD was induced in (C.B-20 X B10.D2)F1 [H-2d, H-40b X H-2d,H-40b] recipients after injection of B10.D2-primed C.B-20 donor cells. GVHD was indicated by the histologic appearance of tissue sections from C.B-20----F1 livers, target organs of GVHD, which showed a marked mononuclear cell infiltrate around the portal tracts and central veins. In addition, splenic lymphocytes from these mice had altered CD4/CD8 ratios and were unable to respond to the polyclonal activators Con A and LPS. The mitogen unresponsiveness was at least partially due to the presence of a suppressor cell, because proliferation of normal spleen cells to Con A and LPS was suppressed upon addition of C.B-20----F1 spleen cells. Further immune dysfunction was evident by the inability of T cells from mice with GVHD to generate a CTL response to H-2 alloantigens. Addition of C.B-20----F1 spleen cells to F1 responder cells at the induction of culture did not prevent generation of CTL, indicating that a suppressor cell was not responsible for the lack of CTL activity. In this setting of GVHD, F1 recipients were able to reject BCL1 upon adoptive transfer of C.B-20 anti-BCL1 effector cells. These data indicate that GVHD-induced immune dysfunction does not inhibit the activity of antileukemia T cells.     

Histological alterations in the livers of Cx43-deficient mice submitted to a cholestasis model

Gap junction intercellular communication capacity and connexin expression are reportedly involved in cell proliferation. To understand the participation of connexins in biliary duct hyperplasia, a cholestasis model was applied to mice with heterologous deletion of Gja 1, the connexin 43 (Cx43) gene. Heterozygous (Cx43+/-) knockout (KO) and wild-type mice (Cx43+/+) (WT) were submitted to bile duct ligation and euthanized at different time points (48 h, 7 days, and 14 days) after surgery. After euthanasia, the macroscopic and microscopic liver alterations were examined. A histomorphometric study of the livers was performed. For this purpose, a grid containing 100 points was applied to each liver section. The volumetric fraction of bile ducts, hepatocytes, arterioles, and terminal hepatic vein were quantified. Cell proliferation was also quantified by western blot PCNA. High mortality was observed in both genotypes. The heterologous deletion of Cx43 did not affect the biliary duct hyperplasia or most of the other parameters analyzed; however, the Cx43-deficient mice showed decrease in hepatic vein angiogenesis in comparison with the wild-type mice 48 h after surgery. In conclusion, our results indicate that the Cx43 gene heterologous deletion does not affect the biliary duct hyperplasia; on the other hand, connexin 43 deficiencies do affect the hepatic vein angiogenesis, although other studies to understand the details of this influence will be necessary.     

Mast cell responses to Hymenolepis microstoma infection in mice

Murine mast cells (MC) responded strongly to Hymenolepis microstoma infection. Starting day 7 postinfection (PI) and continuing until the end of the experiment (35 days PI), significantly larger numbers of MC were present in both the duodenum and bile duct of infected mice than in uninfected controls. In animals challenged with 5 cysticercoids 2 wk after primary infection, the MC response in the duodenum, but not in the bile duct, was of even greater degree than in naive hosts. The majority of MC in the duodenum of infected and challenged mice were intraepithelial mucosal MC, whereas in the bile duct the majority were connective tissue MC. Hypertrophy of the duodenal submucosa and of the bile duct wall was noticeable in all infected and challenged hosts. Worms in primary infections were not affected by the host response, but none of the worms in the challenge dose became established. It is postulated that the type of MC involved in specific immune response of the host is the intraepithelial MC, whereas the cell type participating in general inflammatory events is the connective tissue MC.