Inhibition of leukotriene B4-receptor interaction suppresses eosinophil infiltration and disease pathology in a murine model of experimental allergic encephalomyelitis.

Leukotriene B4 (LTB4) is a chemotactic and cell-activating factor present at inflammatory sites in a variety of autoimmune diseases including multiple sclerosis (MS). In this study, we used a murine model of MS, experimental allergic encephalomyelitis (EAE), to assess the potential role of LTB4 on cell infiltration and paralysis. Injection of encephalogenic T cells into naive animals induced paralysis and weight loss that was completely inhibited by treatment with the selective LTB4 receptor antagonist CP-105,696 (ED50= 8.6 mg/kg orally). Although migration of lymphocytes into the central nervous system was unaffected, the efficacious effects of CP-105,696 correlated with up to a 97% decrease in eosinophil infiltration into the lower spinal cord as determined by light and electron microscopy and quantitated by levels of the specific enzyme marker eosinophil peroxidase. These results demonstrate that eosinophil recruitment in EAE is dependent on LTB4 receptor ligation and further reveal a previously unrecognized role for eosinophils in the pathogenesis of this disease.

Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3.

Chemokines are essential mediators of normal leukocyte trafficking as well as of leukocyte recruitment during inflammation. We describe here a novel non-ELR CXC chemokine identified through sequence analysis of cDNAs derived from cytokine-activated primary human astrocytes. This novel chemokine, referred to as I-TAC (interferon-inducible T cell alpha chemoattractant), is regulated by interferon (IFN) and has potent chemoattractant activity for interleukin (IL)-2-activated T cells, but not for freshly isolated unstimulated T cells, neutrophils, or monocytes. I-TAC interacts selectively with CXCR3, which is the receptor for two other IFN-inducible chemokines, the IFN-gamma-inducible 10-kD protein (IP-10) and IFN-gamma- induced human monokine (HuMig), but with a significantly higher affinity. In addition, higher potency and efficacy of I-TAC over IP-10 and HuMig is demonstrated by transient mobilization of intracellular calcium as well as chemotactic migration in both activated T cells and transfected cell lines expressing CXCR3. Stimulation of astrocytes with IFN-gamma and IL-1 together results in an approximately 400,000-fold increase in I-TAC mRNA expression, whereas stimulating monocytes with either of the cytokines alone or in combination results in only a 100-fold increase in the level of I-TAC transcript. Moderate expression is also observed in pancreas, lung, thymus, and spleen. The high level of expression in IFN- and IL-1-stimulated astrocytes suggests that I-TAC could be a major chemoattractant for effector T cells involved in the pathophysiology of neuroinflammatory disorders, although I-TAC may also play a role in the migration of activated T cells during IFN-dominated immune responses.

Identification of C-C chemokine receptor 1 (CCR1) as the monocyte hemofiltrate C-C chemokine (HCC)-1 receptor.

Hemofiltrate C-C chemokine (HCC)-1 is a recently cloned C-C chemokine that is structurally similar to macrophage inflammatory protein (MIP)-1alpha. Unlike most chemokines, it is constitutively secreted by tissues and is present at high concentrations in normal human plasma. Also atypical for chemokines, HCC-1 is reported not to be chemotactic for leukocytes. In this paper, we have investigated the chemokine receptor usage and downstream signaling pathways of HCC-1. Cross-desensitization experiments using THP-1 cells suggested that HCC-1 and MIP-1alpha activated the same receptor. Experiments using a panel of cloned chemokine receptors revealed that HCC-1 specifically activated C-C chemokine receptor (CCR)1, but not closely related receptors, including CCR5. HCC-1 competed with MIP-1alpha for binding to CCR1-transfected cells, but with a markedly reduced affinity (IC50 = 93 nM versus 1.3 nM for MIP-1alpha). Similarly, HCC-1 was less potent than MIP-1alpha in inducing inhibition of adenylyl cyclase in CCR1-transfected cells. HCC-1 induced chemotaxis of freshly isolated human monocytes, THP-1 cells, and CCR1-transfected cells, and the optimal concentration for cell migration (100 nM) was approximately 100-fold lower than that of MIP-1alpha (1 nM). These data demonstrate that HCC-1 is a chemoattractant and identify CCR1 as a functional HCC-1 receptor on human monocytes.

In vivo properties of monocyte chemoattractant protein-1.

Monocyte chemoattractant protein-1 (MCP-1) attracts monocytes, memory T lymphocytes, and natural killer (NK) cells in vitro. Its expression has been documented in disorders characterized by mononuclear cell infiltrates, suggesting that it may contribute to the inflammatory component of such diseases as atherosclerosis, multiple sclerosis, or rheumatoid arthritis. To prove a causal association, the in vivo properties of MCP-1 must be understood. Several lines of transgenic mice have been constructed to address this question. A transgenic line in which MCP-1 expression is controlled by the MMTV-LTR expressed high levels of MCP-1 in multiple organs but showed no evidence for monocyte infiltration. Instead, these mice were more susceptible to infection by the intracellular pathogens, Listeria monocytogenes and Mycobacterium tuberculosis. These mice had high serum levels of MCP-1, suggesting that their circulating monocytes may have been desensitized or that MCP-1 stimulated a Th2-dominant response. In contrast, another model in which MCP-1 expression was controlled by the insulin promoter demonstrated a monocytic infiltrate in pancreatic islets. These results indicate that MCP-1 expression at low levels in an anatomically confined area results in monocyte infiltration, suggesting that when properly expressed, MCP-1's in vitro properties are reproduced in vivo. This justifies the examination of MCP-1-deficient mice in disease models in order to explore MCP-1's role in pathogenesis.

The protective effects of the thromboxane synthetase inhibitor Dazmegrel on nephrotoxicity in cyclosporine-treated rats.

Renal vasoconstriction has been implicated as a major contributing factor for the nephrotoxic effects of cyclosporine. In an attempt to assess the relative contribution of thromboxane (Tx), the effects of coadministering CsA with the selective Tx synthetase inhibitor, Dazmegrel (DAZ) (Pfizer, Inc.), were determined. Rats were treated orally with 50 mg/kg of DAZ plus 50 mg/kg of CsA and various indicators of nephrotoxicity and efficacy were assessed. Animals treated with CsA + DAZ had a normalization of renal TxB2 synthesis as compared with animals treated with CsA alone (160 vs. 338 pg/ml). Kidney proximal tubule damage following CsA treatment alone was also reduced in animals coadministered DAZ, as indicated by reduced urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) (9.7 vs. 14.1 U/g creatinine) and by histological examination of kidney sections. However, DAZ did not affect blood levels of CsA nor its efficacious activity in a model of experimental allergic encephalomyelitis (EAE). These studies suggest a major role of elevated thromboxane production in the acute nephrotoxic effects of CsA and demonstrate a reduction in this toxicity by DAZ without altering CsA's efficacious activity.

Differential expression of chemokines and chemokine receptors shapes the inflammatory response in rejecting human liver transplants.

BACKGROUND: Graft rejection after liver transplantation is associated with a lymphocytic infiltrate, the nature of which will be determined by, among various factors, the local activity of chemokines that attract particular subsets of effector cells to the graft. METHODS: The expression of chemokines and receptors in human liver allografts was studied by immunohistochemistry of tissue and flow cytometry of blood and liver-derived lymphocytes. Receptor function was assessed with in vitro chemotaxis. RESULTS: We report increased expression of chemokine receptors CXCR3, CXCR4, and CCR5 on circulating and graft-infiltrating lymphocytes after liver transplantation. Liver-derived T cells responded to the ligands for these receptors in vitro, which suggests that the receptors are functionally active. The chemokine ligands for these receptors were detected in rejecting allografts. CXCR3 ligands interferon-inducible protein 10 and monokine-induced by gamma interferon were detected on sinusoidal endothelium and interferon-inducible T-cell alpha chemoattractant was detected on portal and hepatic vascular endothelium, whereas the CXCR4 ligand, stromal-derived factor (SDF), was restricted to biliary epithelium. CCR5 ligands have previously been shown on portal endothelium. An in vitro model of T-cell alloactivation demonstrated a similar pattern of expression of functional CXCR3, CXCR4, and CCR5 on T cells. Increased expression of chemokine receptors, especially CCR3 and CCR5, was associated with redistribution of activated Kupffer cells in rejecting grafts. CONCLUSIONS: The patterns of chemokine expression in liver allografts during rejection suggest that the recruitment and positioning of lymphocytes is mediated by specific chemokines. Although ligands for the receptors CXCR3 and CCR5 are important for recruitment, the restriction of SDF to bile ducts suggests that CXCR4 may be involved in the retention of alloactivated lymphocytes at sites of graft damage.

The CC chemokine MIP-1alpha induces a selective monocyte infiltration following intradermal injection into nonhuman primates.

The in vitro chemotactic activity of chemokines have been well documented. However, study of their in vivo effects where components of rolling, adherence and diapedesis are pre-requisites to leukocyte infiltration have not been examined in higher species. In this study, we examined the biological activity of the CC chemokine, MIP-1alpha, in rhesus monkeys. Following an intradermal injection, a significant cellular infiltrate and an increase in the number of inflamed vessels were observed. This response peaked at 24 h and was sustained for up to 48 hours after injection. Phenotypically, the specific infiltrate consisted exclusively of CD68+ monocytes with no increase in other cell types over the saline injected controls. These studies represent the first examination of the in vivo effects of MIP-1alpha in higher species and indicate that MIP-1alpha is a selective monocyte recruiting agent in vivo.

Promoting social health in northern Saskatchewan.

In response to concern about social health problems in Northern Saskatchewan, a Working Group on Social Health was established in 1989 in the Research and Development Committee of Northern Medical Services. The Group formulated a concept of mental health in social terms; found and interpreted indicators of the extent of social health problems; identified major determinants of social health problems, barriers to effective coping and problems in providing adequate support and services; and identified strategies and program models that could be more effective in promoting social health in this region. Indicators of problems and underlying determinants are discussed, along with strategies for change. These strategies are based on a community development model, and incorporate innovation and reaffirmation of values and ways that have traditionally given people strength.

An agonist antibody specific for CD40 induces dendritic cell maturation and promotes autologous anti-tumour T-cell responses in an in vitro mixed autologous tumour cell/lymph node cell model.

CD40-mediated interactions play an important role in the response to a variety of diseases, including cancer. Engagement of CD40 on antigen-presenting cells, namely dendritic cells (DC), by CD40L leads to maturation and up-regulation of co-stimulatory molecules B7.1 and B7.2 (CD80 and CD86). These molecules are requisite to subsequent antigen-specific activation of T cells. T-cell activation is a critical aspect of specific anti-tumour immune responses that have become the focus of a variety of cancer immunotherapy approaches. Clinical trials involving immunologic interventions have shown clinical responses confirming that the immune system can be harnessed for the treatment of cancer. However, the clinical response rate has been low, signifying the need for new immunotherapeutic strategies. To this end, an agonist antibody specific for CD40, CP-870,893, has been developed. A fully autologous mixed tumour cell/lymph node cell model was utilized to demonstrate that CP-870,893 promotes the responsiveness of lymph node-derived T cells to autologous tumour. Specifically, T cells from the tumour-draining lymph nodes are not responsive to autologous tumour cells; however, in the presence of CP-870,893, this unresponsiveness is reversed, as indicated by lymph node cell proliferation and cytokine secretion. Monocyte-derived DC treated with CP-870,893 consistently display a mature phenotype: up-regulation of CD80, CD83, CD86 and HLA-DR expression, increased Mip1alpha and IL-12 secretion, and the loss of exogenous antigen-presenting capability subsequent to treatment with the antibody. These data indicate that CP-870,893 binds to and activates DC, ultimately driving a specific anti-tumour T-cell response.

Enhanced antibacterial resistance in neutropenic mice treated with human recombinant interleukin-1 beta.

Human Recombinant IL-1 was investigated for its ability to increase non-specific resistance to Staphylococcus aureus in neutropenic mice. Mice, rendered neutropenic with cyclophosphamide and then administered IL-1 intraperitoneally, demonstrated enhanced resistance to subsequent challenge with S. aureus as measured by increased survival and bacterial clearance. No protective effects were observed with heat inactivated IL-1. Efficacy was observed only when both IL-1 and the subsequent bacterial challenge were administered into the same site. Despite the observed protective effects, animals treated with IL-1 did not have increased numbers of blood leukocytes or peritoneal phagocytes prior to infection or at the times coincident with bacterial clearance. Based upon these observations, enhanced activity of resident macrophages may be responsible for the protective effects observed in IL-1 treated mice.

Intracellular accumulation of azithromycin by cultured human fibroblasts.

Azithromycin was shown to achieve high concentrations in human skin fibroblasts. Intracellular penetration occurred rapidly (10 micrograms/mg of cellular protein after 3 h) and then increased progressively over a 3-day period; azithromycin accumulated up to 21 times more than erythromycin (61.1 versus 2.9 micrograms/mg of protein). Uptake was dependent on the extracellular concentration, was inhibited at 4 degrees C, did not occur in nonviable cells, and was reduced by a low pH. Intracellular accumulation was not affected by the metabolic inhibitor 2,4-dinitrophenol or sodium fluoride or by the nucleoside transport inhibitor 2-chloradenosine. Once concentrated in cells, azithromycin remained intracellular and was released slowly in the absence of extracellular drug, compared with erythromycin (17 versus 78% released after 1 h). After 48 h of incubation in drug-free medium, 27% of the initial amount of azithromycin remained cell associated. The release of azithromycin was not affected by various monokines reported to stimulate fibroblasts (interleukin-1 or tumor necrosis factor) or by exposure to bacteria. Incubation of azithromycin-loaded fibroblasts with human polymorphonuclear leukocytes resulted in a higher intracellular accumulation of azithromycin in polymorphonuclear leukocytes than in cells incubated with free nonintracellular azithromycin for the same time (8.3 versus 2.2 micrograms/ml after 2 h), suggesting a more efficient or rapid uptake through cell-to-cell interaction. The widespread distribution of fibroblasts in tissues suggests a potential for these cells, and possibly other lysosome-containing tissue cells, to serve as a reservoir for azithromycin, slowly releasing it for activity against extracellular organisms at sites of infection and passing it to phagocytes for activity against intracellular pathogens and potential transport to sites of infection.

In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection.

Azithromycin, a novel azalide antibiotic, concentrated in human and mouse polymorphonuclear leukocytes (PMNs), murine peritoneal macrophages, and mouse and rat alveolar macrophages, attaining intracellular concentrations up to 226 times the external concentration in vitro. In murine peritoneal macrophages, azithromycin achieved concentration gradients (internal to external) up to 26 times higher than erythromycin. The cellular uptake of azithromycin was dependent on temperature, viability, and pH and was decreased by 2,4-dinitrophenol. Azithromycin did not decrease phagocyte-mediated bactericidal activity or affect PMN or macrophage oxidative burst activity (H2O2 release or Nitro Blue Tetrazolium reduction, respectively). Azithromycin remained in cells for several hours, even after extracellular drug was removed. However, its release was significantly enhanced by phagocytosis of Staphylococcus aureus (82 versus 23% by 1.5 h). In vivo, 0.05 micrograms of azithromycin was found in peritoneal fluids of mice 20 h after oral treatment with a dose of 50 mg/kg. Following caseinate-induced PMN infiltration, there was a sixfold increase in peritoneal cavity azithromycin to 0.32 micrograms, most of which was intracellular. Therefore, the uptake, transport, and later release of azithromycin by these cells demonstrate that phagocytes may deliver active drug to sites of infection.

Activity of trovafloxacin (CP-99,219) against Legionella isolates: in vitro activity, intracellular accumulation and killing in macrophages, and pharmacokinetics and treatment of guinea pigs with L. pneumophila pneumonia.

The activity of trovafloxacin against 22 clinical Legionella isolates was determined by broth microdilution susceptibility testing. The trovafloxacin concentration required to inhibit 90% of strains tested was < or = 0.004 micrograms/ml, in contrast to 0.032 micrograms/ml for ofloxacin. In guinea pig alveolar macrophages, trovafloxacin achieved intracellular levels up to 28-fold over the extracellular concentration, which was similar to the levels obtained with erythromycin. Trovafloxacin (0.25 micrograms/ml) reduced bacterial counts of two L. pneumophila strains grown in guinea pig alveolar macrophages by > 2 log10 CFU/ml, without regrowth, under drug-free conditions over a 3-day period; trovafloxacin was significantly more active than ofloxacin or erythromycin (0.25 to 1 microgram/ml) in this assay. Single-dose (10 mg of prodrug CP-116,517-27 per kg of body weight given intraperitoneally [i.p.], equivalent to 7.5 mg of trovafloxacin per kg) pharmacokinetic studies performed in guinea pigs with L. pneumophila pneumonia revealed peak serum and lung trovafloxacin levels to be 3.8 micrograms/ml and 5.0 micrograms/g, respectively, at 0.5 h and 4.2 micrograms/ml and 2.9 micrograms/g, respectively, at 1 h. Administration of a lower prodrug dose (1.4 mg of trovafloxacin equivalent per kg i.p.) gave levels in lung and serum of 0.4 microgram/g and 0.4 microgram/ml, respectively, 1 h after drug administration. The terminal half-lives of elimination from serum and lung were 0.8 and 1.1 h, respectively. All 15 infected guinea pigs treated for 5 days with CP-116,517-27 once daily (10 mg/kg/day i.p., equivalent to 7.5 mg of trovafloxacin per kg/day) survived for 10 days after antimicrobial therapy, as did all 15 guinea pigs treated with ofloxacin once daily (10 mg/kg/day i.p.) for 5 days. None of 13 animals treated with saline survived. In a second experiment with animals, trovafloxacin (1.4 mg/kg/day i.p. for 5 days) protected all 16 guinea pigs from death, whereas all 15 animals treated with saline died. Trovafloxacin is an effective antimicrobial agent against Legionella in vitro and in vivo, with the ability to concentrate in macrophages and kill intracellular organisms.

Chemokine networks in vivo: involvement of C-X-C and C-C chemokines in neutrophil extravasation in vivo in response to TNF-alpha.

In this study, we have evaluated the role of specific chemotactic cytokines in leukocyte recruitment to s.c. tissue in response to TNF-alpha in vivo. Injection of TNF-alpha into s.c. air pouches led to a rapid, transient accumulation of leukocytes. Maximal accumulation of leukocytes in the air pouch was observed at between 2 and 4 h after injection of TNF-alpha. The cellular exudate comprised predominantly neutrophils, with smaller numbers of eosinophils and mononuclear phagocytes also being recruited. However, lymphocyte recruitment was not observed. TNF-alpha injection induced a time-dependent increase in the levels of immunoreactive macrophage inflammatory protein (MIP)-2, MIP-1alpha, and JE in the pouch exudate as well as increased steady-state mRNA levels of KC, MIP-2, MIP-1alpha, and JE in the tissue lining the s.c. pouch and of MIP-2, MIP-1alpha, and JE in the exudate cell population. Passive immunization with specific Abs directed against each of these chemokines significantly inhibited the accumulation of neutrophils, mononuclear phagocytes, and eosinophils in response to TNF-alpha. Taken together, these data demonstrate the existence of a chemokine network in vivo involving at least four individual chemokines that regulates recruitment of the major peripheral blood granulocytes and mononuclear phagocytes to s.c. sites during acute inflammation. To our knowledge, these data are also the first demonstration that the C-C chemokine JE is involved in neutrophil recruitment in a physiologic system in vivo.

Inhibition of functional T cell priming and contact hypersensitivity responses by treatment with anti-secondary lymphoid chemokine antibody during hapten sensitization.

Recent studies have suggested a pivotal role for secondary lymphoid chemokine (SLC) in directing dendritic cell trafficking from peripheral to lymphoid tissues. As an extension of these studies, we examined the consequences of anti-SLC Ab treatment during Ag priming on T cell function in an inflammatory response. We used a model of T cell-mediated inflammation, contact hypersensitivity (CHS), where priming of the effector T cells is dependent upon epidermal dendritic cell, Langerhans cells, and migration from the hapten sensitization site in the skin to draining lymph nodes. A single injection of anti-SLC Ab given at the time of sensitization with FITC inhibited Langerhans cell migration into draining lymph nodes for at least 3 days. The CHS response to hapten challenge was inhibited by anti-SLC Ab treatment in a dose-dependent manner. Despite the inhibition of CHS, T cells producing IFN-gamma following in vitro stimulation with anti-CD3 mAb or with hapten-labeled cells were present in the skin-draining lymph nodes of mice treated with anti-SLC Ab during hapten sensitization. These T cells were unable, however, to passively transfer CHS to naive recipients. Animals treated with anti-SLC Ab during hapten sensitization were not tolerant to subsequent sensitization and challenge with the hapten. In addition, anti-SLC Ab did not inhibit CHS responses when given at the time of hapten challenge. These results indicate an important role for SLC during sensitization for CHS and suggest a strategy to circumvent functional T cell priming for inflammatory responses through administration of an Ab inhibiting dendritic cell trafficking.

Early eosinophil infiltration into the optic nerve of mice with experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis is a murine model of preclinical autoimmune disease that has pathologic similarities to multiple sclerosis (MS). Although CD4+ T cells have been shown to play a crucial role in the development of disease, we recently demonstrated a link between the development of paralysis and eosinophil infiltration into the spinal cord. As such, CD4+ cells may initiate disease, but eosinophils may be the actual effector cells responsible for causing damage to myelin and causing paralysis. Because MS patients sometimes experience early visual problems, ie, optic neuritis, we explored whether an early eosinophil infiltrate was also observed in the optic nerves of SJL mice after the passive transfer of encephalitogenic T cells. Seven days after the passive transfer of myelin basic protein (MBP)-reactive T cell blasts, we observed a significant infiltration of eosinophils into the optic nerves of the mice. This infiltration persisted during the early phases of paralysis, then declined to baseline values by the peak of limb paralysis on Day 10, and remained at baseline during the remission phase of the disease. Remyelination of optic nerves was observed at this time. These results suggest that eosinophil infiltration into the optic nerve is one of the earliest events occurring after the passive transfer of encephalitogenic T cells in murine experimental allergic encephalomyelitis.

Ccr2 regulates the level of MCP-1/CCL2 in vitro and at inflammatory sites and controls T cell activation in response to alloantigen.

CCR2, and its principle ligand MCP-1/CCL2, have been well documented for their ability to induce monocyte infiltration and promote the pathogenesis of rheumatoid arthritis and atherosclerosis. In order to assess additional roles for CCR2, we inserted allogeneic implants into CCR2-/- and MCP-1-/- mice and characterized T cell responses and the regulatory role of CCR2 on MCP-1 expression. The results demonstrate a marked decrease in lymphocyte infiltration in both CCR2-/- and MCP-1-/- animals. In contrast, IL-12 and CTL function were only suppressed in CCR2-/- animals. Further, whereas MCP-1 was only transiently elevated in the inflammatory fluid of WT animals, levels were sustained within the implants (5000pg/ml; >8 days) and serum (243pg/ml) of CCR2-/- mice. Higher levels of MCP-1 were also observed in the culture supernatants of CCR2-/- macrophages as compared to WT cells despite no difference in mRNA levels. Evidence that MCP-1 levels are regulated by receptor binding and internalization was suggested by its rapid decline when added to WT macrophages at 37 degrees C but not 4 degrees C. These studies indicate that CCR2 plays an important role in regulating T cell responses and controlling the level of MCP-1 at inflammatory sites.

Phenotypic characterization and analysis of allogeneic T cell responses in ZAP-70 dominant negative transgenic mice.

Antigen stimulation of T cells results in a series of biochemical events including the interaction of both SH2 domains of ZAP-70 with phosphorylated ITAMS on the T cell receptor. In order to study the physiological relevance of decreasing native ZAP-70-SH2 interaction in vivo, we generated transgenic mice expressing a T cell-specific, dominant negative form of ZAP-70 consisting of only the tandem SH2 domains (ZAP-NC). Phenotypically, these animals had a comparable distribution of lymphocyte subsets in the thymus and spleen compared with the wild-type (WT) controls. However, examination of peripheral blood revealed a slow but progressive decrease in the number of lymphocytes, particularly CD4+ cells, with age (17% reduction by 3 months, 58% reduction by 6 months). Allogeneic responses were then evaluated in vitro as well as in vivo using a subcutaneous sponge matrix implant. Although spleen cells cultured for 4 days in vitro with alloantigen developed normal functional responses, allogeneic responses generated in vivo within a subcutaneous sponge matrix were impaired. This was characterized by a depression in cytotoxic T lymphocyte (CTL) activity, a 82% reduction in the frequency of helper T cells, and a 78% reduction in the capacity of sponge-infiltrating lymphocytes to produce IL-2 in response to secondary antigen stimulation. These results indicate that although overt lymphocyte development and in vitro function were unremarkable, expression of a truncated ZAP-70 affected the in vivo survival of peripheral lymphocytes and altered the in vivo generation of functional activity to alloantigen.

Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1.

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that attracts monocytes and T lymphocytes in vitro; however, its in vivo functions are poorly understood. To address this question, we constructed transgenic mice expressing MCP-1 controlled by an insulin promoter. These mice developed a chronic insulitic infiltrate composed of F4/80+ monocytes with minor populations of CD4+, CD8+, and B220+ cells. Despite persistent transgene expression, the insulitis never progressed, and blood glucose levels remained normal. Thus, MCP-1 alone is sufficient to elicit a monocytic infiltrate, but not to activate elicited cells. These results differ from those obtained with another transgenic model using the mouse mammary tumor virus long terminal repeat, in which mice expressed substantial MCP-1 in several organs but had no infiltrates. However, mice expressing both transgenes had minimal insulitis, indicating that high systemic levels of MCP-1 prevented monocytes from responding to local MCP-1. Thus, the ability of MCP-1 to elicit monocytic infiltration depends on its being expressed at low levels in an anatomically restricted area.

High level monocyte chemoattractant protein-1 expression in transgenic mice increases their susceptibility to intracellular pathogens.

We have constructed transgenic mice in which the mouse mammary tumor virus long terminal repeat controls the expression of murine monocyte chemoattractant protein-1 (MCP-1). Several independently derived lines of transgenic mice constitutively expressed MCP-1 protein in a variety of organs. Protein extracts from these organs had substantial in vitro monocyte chemoattractant activity that was neutralized by an anti-MCP-1 Ab, indicating that transgenic MCP-1 protein is biologically active. However, no transgenic mouse at any age displayed monocyte infiltrates in MCP-1-expressing organs. Two transgenic lines had circulating MCP-1 levels of 13 to 26 ng/ml, which is a concentration sufficient to induce maximal monocyte chemotaxis in vitro. These transgenic lines showed a 1 to 1.5 log greater sensitivity to infection with Listeria monocytogenes and Mycobacterium tuberculosis. A third transgenic line had lower serum levels of MCP-1 and was resistant to L. monocytogenes. The results suggest that this transgenic model is one of monocyte nonresponsiveness to locally produced MCP-1 due to either receptor desensitization or neutralization of a chemoattractant gradient by high systemic concentrations of MCP-1. Regardless of the mechanism, the data indicate that constitutively high levels of MCP-1 expression do not induce monocytic infiltrates, and that MCP-1 is involved in the host response to intracellular pathogens.