Manfred Kopf

OX40-deficient mice are defective in Th cell proliferation but are competent in generating B cell and CTL Responses after virus infection.

OX40, a member of the TNF receptor superfamily, is expressed on activated T cells and implicated in stimulation of T cells and T-dependent humoral responses. We generated OX40-/- mice and found that the formation of extrafollicular plasma cells, germinal centers, and antibody responses was independent of OX40. After infection with LCMV and influenza virus, OX40-/- mice retain primary and memory cytotoxic T cell responses with normal expansion and decline of specific CTL. In contrast, CD4+ T cell proliferation and the number of IFN-gamma-producing CD4+ T cells were reduced in OX40-/- mice. Moreover, the number of CD4+ T cells infiltrating the lungs of influenza virus-infected OX40-/- mice was reduced. These results define a unique role of OX40 in the generation of optimal CD4+ T cell responses in vivo.

Interleukin-6 Gene-Deficient Mice Show Impaired Defense against Pneumococcal Pneumonia

Induction of pneumonia in C57Bl/6 mice by intranasal inoculation with 10(6) cfu of Streptococcus pneumoniae resulted in sustained expression of interleukin (IL)-6 mRNA in lungs and increases in lung and plasma IL-6 concentrations. In IL-6-deficient (IL-6-/-) mice, pneumonia was associated with higher lung levels of the proinflammatory cytokines tumor necrosis factor-alpha, IL-1beta, and interferon-gamma and of the antiinflammatory cytokine IL-10 than in wild type (IL-6+/+) mice (all P < .05). Also, the plasma concentrations of soluble tumor necrosis factor receptors were higher in IL-6-/- mice (P < .05), while the acute-phase protein response was strongly attenuated (P < .01). Lungs harvested from IL-6-/- mice 40 h after inoculation contained more S. pneumoniae colonies (P < .05). IL-6-/- mice died significantly earlier from pneumococcal pneumonia than did IL-6+/+ mice (P < .05). During pneumococcal pneumonia, IL-6 down-regulates the activation of the cytokine network in the lung and contributes to host defense.

IL-6-deficient mice resist myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is induced by immunization with myelin components including myelin oligodendrocyte glycoprotein (MOG). Myelin‐specific Th1 cells enter the central nervous system (CNS) via binding of very late antigen 4 (VLA‐4) to the endothelial vascular cell adhesion molecule 1 (VCAM‐1). In the present study, mice with a homologous disruption of the gene encoding IL‐6 are found to be resistant to MOG‐induced EAE as evidenced by absence of clinical symptoms, minimal infiltration of CD3+ T cells and monocytes into the CNS and lack of demyelination. The failure to induce EAE in IL‐6−u2009/u2009− mice is not due to the absence of priming, since lymphocytes of immunized IL‐6−u2009/u2009− mice proliferate in response to MOG and produce pro‐inflammatory cytokines including IL‐2 and IFN‐γ. However, in MOG‐immunized IL‐6−u2009/u2009− mice, serum anti‐MOG antibody titers were found to be drastically reduced. This observation is unlikely to be responsible for resistance to EAE, because B cell‐deficient (μMT) mice proved to be fully susceptible to the disease. A striking difference between MOG‐immunized wild‐type (wt) and IL‐6−u2009/u2009− mice was the expression of endothelial VCAM‐1 and ICAM‐1, which were dramatically up‐regulated in the CNS in wt but not in IL‐6−u2009/u2009− mice. Taking into account recent studies on the role of VCAM‐1 in the entry of Th1 cells into the CNS, the absence of VCAM‐1 on endothelial cells in IL‐6−u2009/u2009− mice may explain their resistance to EAE.

Mouse Eotaxin expression parallels eosinophil accumulation during lung allergic inflammation but it is not restricted to a Th2-type response

A model of lung eosinophilia based on the repeated exposure of mice to aerosolized OVA has been used to identify C-C chemokine genes expressed at stages of massive eosinophil infiltration. We describe the identification and cloning of a cDNA that encodes a mouse C-C chemokine with 68% amino acid identity to guinea pig Eotaxin. The recombinant protein encoded by this gene displays potent and specific chemotactic activity for eosinophils, both in vivo and in vitro. Its mRNA levels parallel the kinetics of eosinophil accumulation in the lung during the experimentally induced eosinophilia and it is mainly produced by type I alveolar epithelial cells. The mRNA expression of mouse Eotaxin is not restricted to Th2 T cells in vitro and is independent of the development of a Th2-type response during N. brasiliensis infection, in vivo.

Distinct kinetics of cytokine production and cytolysis in effector and memory T cells after viral infection

In the present study, naive Tu2004cells were compared with in vivo generated effector and memory Tu2004cells expressing the same TCR specific for lymphocytic choriomeningitis virus. Upon restimulation in vitro, the same minimal concentrations of the full agonist peptide p33 and also of weak and partial agonist peptides were required for proliferation of naive, effector and memory Tu2004cells, indicating no difference in threshold of activation. However, activation kinetics were distinct. While effector cytotoxic Tu2004cells exhibited immediate ex vivo lytic effector function, naive and memory Tu2004cells required 12u2004h and more exposure to antigen to developlytic activity. However, both effector and memory Tu2004cells contained IFN‐γ mRNA in vivo and required less than 3u2004h for secretion of cytokines upon restimulation in vitro. In contrast, naive Tu2004cells did not contain IFN‐γ mRNA and required more than 12u2004h for cytokine secretion. Our results show that memory Tu2004cells exhibit a unique phenotype in that they produce cytokines and commit to proliferation as rapidly as effector cells, whereas they resemble naive Tu2004cells in the time requirement for development of cytolytic function.

Differences between IL-4Rα-deficient and IL-4-deficient mice reveal a role for IL-13 in the regulation of Th2 responses

Allergens and infections with parasitic helminths preferentially induced Th2 immune responses associated with elevated levels of serum immunoglobulin E (IgE) and expansion of eosinophils and mast cells. Interleukin-4 (IL-4) is a key cytokine in the differentiation of naive CD4+ T cells into Th2 cells, which produce a panel of cytokines including IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 [1] and have been shown to trigger recovery from gastrointestinal nematodes [2]. Nonetheless, mice deficient for IL-4 have been shown to develop residual Th2 responses [3-5] and can expel the nematode Nippostrongylus brasiliensis [6], suggesting that there is a functional equivalent of IL-4 in these processes. IL-13 is a cytokine that shares some, but not all, biological activities with IL-4 [7,8]. There is now compelling evidence that IL-4 and IL-13 share receptor components, including IL-4R alpha and IL-13R alpha 1 [9]. In order to dissect the roles of IL-4 and IL-13 in the regulation of Th2 cells and in the response to nematode infections, we looked for differences between mice deficient for either the IL-4 gene or the IL-4R alpha gene. Unlike IL-4, IL-4R alpha was required for control of N. brasiliensis, and Th2 development during infection--as characterized by cytokine production, GATA-3 and surface CD30 expression--was more severely affected in IL-4R alpha-/- mice than in IL-4-/- mice. Injection of recombinant IL-13 induced worm expulsion in otherwise incompetent RAG2-/- mice. Our results suggest that IL-13 regulates Th2 responses to nematode infection and requires IL-4R alpha.

Interleukin-4 Causes Susceptibility to Invasive Pulmonary Aspergillosis through Suppression of Protective Type I Responses

Aspergillus fumigatus, an opportunistic fungal pathogen, causes multiple allergic and nonallergic airway diseases. Invasive pulmonary aspergillosis (IPA) is a nonallergic, life-threatening disease of immunocompromised patients. In a murine model of IPA, interleukin (IL)-4-deficient (IL-4-/-) BALB/c mice were used to examine the role of IL-4 in lung pathology and immune responses. IL-4-/- mice were more resistant than wild-type mice to infection caused by multiple intranasal injections of viable A. fumigatus conidia. Resistance was associated with decreased lung inflammatory pathology, impaired T helper (Th)-2 responses (including lung eosinophilia), and an IL-12-dependent Th1 response. In contrast, development of host-detrimental antifungal Th2 cells occurred in IL-12-/- and interferon-gamma-/- mice and in IL-4-/- mice when subjected to IL-12 neutralization. These results demonstrate that IL-4 renders mice susceptible to infection with A. fumigatus by inhibition of protective Th1 responses. IL-4 appears to have a distinct role in the pathogenesis of allergic and nonallergic lung diseases caused by the fungus.

The antigen dose determines T helper subset development by regulation of CD40 ligand

Although the amount of antigen and the strength of T cell stimulation have been suggested to regulate Th1 vs. Th2 polarization, it remains unclear how the antigen dose and the strength of signal is detected by the T cell and translated into differential cytokine production. Using co‐cultures of dendritic cells (DC) and ovalbumin (OVA)‐specific CD4+ T cells obtained from RAG‐2–u2009/u2009– DO11.10 mice, we show here that high‐dose antigen induced Th1 development by up‐regulation of CD40 ligand (CD40L), whereas low‐dose antigen stimulation failed to induce CD40L and promoted Th2 development. CD40‐CD40L interaction was essential for IL‐12 production by DC. In the absence, de novo IL‐4 production by T cells and autocrine Th2 development was induced. Furthermore, our results demonstrate that LFA‐1u2009/u2009ICAM interaction promotes Th1 differentiation by lowering the antigen dose required for CD40L up‐regulation. Thus, we propose that (1) peptide‐MHC density and (2) accessory molecules such as LFA‐1 determine T helper polarization by regulation of CD40L.

Immune Responses of IL-4, IL-5, IL-6 Deficient Mice

Cytokines are the principal soluble regulators of the immune system and the hematopoietic system (Paul & Seder 1994). In vivo, cytokines contribute to a complex network of pleiotropic and redundantly acting molecules, which makes them difficult to study. Within the past 4 years, gene targeting has been used to poke holes into this network. The list of mice rendered deficient for cytokines and their receptor is growing (Table I). In contrast to previous ideas, from these efforts we have learned that cytokines do not play unique roles in the development of the immune system and steady state hematopoiesis. Exceptions involve IL-7/IL-7R and TNF-p (LT-a), which dramatically affect early T and B cell precursors and the formation of lymph nodes, respectively (De-Togni et al. 1994, Peschon et al. 1994). However, impairment, or minimally a modulation, of immune and/or infiammatory responses has been shown to occur in each of these cytokine/receptor deficient mice, which clearly demonstrates that no cytokine is dispensable or completely redundant in vivo. This review summarises the effects of either an IL-4, IL-5 or IL-6 gene deletion on the immune response of mice to several distinct kinds of immune stimuli

IL-4 and IL-10 Antagonize IL-12-Mediated Protection Against Acute Vaccinia Virus Infection with a Limited Role of IFN-γ and Nitric Oxide Synthetase 2

Resistance or susceptibility to most infectious diseases is strongly determined by the balance of type 1 vs type 2 cytokines produced during infection. However, for viruses, this scheme may be applicable only to infections with some cytopathic viruses, where IFN-γ is considered as mandatory for host defense with little if any participation of type 2 responses. We studied the role of signature Th1 (IL-12, IFN-γ) and Th2 (IL-4, IL-10) cytokines for immune responses against vaccinia virus (VV). IL-12−/− mice were far more susceptible than IFN-γ−/− mice, and primary CTL responses against VV were absent in IL-12−/− mice but remained intact in IFN-γ−/− mice. Both CD4+ and CD8+ T cells from IL-12−/− mice were unimpaired in IFN-γ production, although CD4+ T cells showed elevated Th2 cytokine responses. Virus replication was impaired in IL-4−/− mice and, even more strikingly, in IL-10−/− mice, which both produced elevated levels of the proinflammatory cytokines IL-1α and IL-6. Thus, IL-4 produced by Th2 cells and IL-10 produced by Th2 cells and probably also by macrophages counteract efficient anti-viral host defense. Surprisingly, NO production, which is considered as a major type 1 effector pathway inhibited by type 2 cytokines, appears to play a limited role against VV, because NO sythetase 2-deficient mice did not show increased viral replication. Thus, our results identify a new role for IL-12 in defense beyond the induction of IFN-γ and show that IL-4 and IL-10 modulate host protective responses to VV.