Macrophages are leukocytes which are present throughout the body. They develop from bone marrow precursors then mature and enter the bloodstream as monocytes. Monocytes are recruited into different tissues where they differentiate into tissue resident macrophages which perform important functions in the immune response. Leishmania is an intracellular protozoan parasite, and it causes leishmaniasis to humans and other mammalian animals. Phagocytic cells such as macrophages and dendritic cells are the host cells to Leishmania and the parasites proliferate inside of these cells. Leishmania major (L. major) causes cutaneous leishmnaiasis, and they develop lesion on the skin. Macrophages widely distribute in the body however, the infection is not observed in macrophages of every tissue. The skin, spleen and lymph nodes are major sites of parasite infection. Several experimental murine models demonstrated that the different susceptibility to L. major depends on the route of infection. Therefore, this site-specific immune response may define the susceptibility to L. major. However, it is still unclear what mechanisms would be involved in the variety immune responses to L. major in the same host. If the sites of infection determine the pathology and immune response induced by the infection then, it is required to know the mechanisms that might be involved in inducing the susceptibility to the infection. Hence, I hypothesized that macrophages may show tissue specific response to L. major, and the responses would relate to the parasite susceptibility.

In the first chapter, L. major infection was investigated using macrophages collected from two sites, lung alveoli and peritoneal cavity, in order to address if the susceptibility to L. major would be variety in macrophages distribute in different sites. The reason is that the infection is not reported in the lung, while peritoneal macrophage (PMphi) are susceptible to L. major and the cells are used to experimental leihsmaniasis in murine model. Then, the collected macrophages were used to this study as an in vitro model. When macrophages were infected to L. major for four hr, 40 ± 5% of PMphi were infected by the parasites, and showed high levels of intracellular parasite. Interestingly, in contrast, fewer infected macrophages were observed in alveolar macrophages (AMphi) (26 ± 1%) and they displayed fewer intracellular parasites than that of PMphi. In addition, at the late phase of infection, 72 hr post-infection (p.i.), infection rate and levels of intracellular parasite of PMphi were higher than those of AMphi. It is thus demonstrated that AMphi and PMphi had different susceptibility to L. major. Next, to evaluate the effect of the activation status on the resistance of macrophages, these macrophages were treated with lipopolisaccaride (LPS), which one of the molecules stimulates macrophages. When macrophages were stimulated with LPS, PMphi showed higher levels of infection rate and held large number of intracellular parasites four hr p.i., compared to untreated control cells. On the contrary, in AMphi, LPS expose did not affect to the rate of infection and intracellular parasite. The regulation phagocytic activity of these macrophages was parasite specific, since identical uptake of latex beads was observed in both types of macrophages. In this chapter, different responses to L. major were observed in two different macrophages, AMphi and PMphi. These results suggest that macrophages distribute different sites would also show various response, and those variations might contribute to the establishment of tissue specific L. major infection.

The fact that macrophages distributed in two sites, lung alveoli and peritoneal cavity, showed different susceptibility to L. major, prompted to examine if macrophage derived factor(s) relate to the different susceptibility of macrophages to the parasites. IFN-y was produced from macrophages, and identified as a cytokine required for not only Th1 cell differentiation but also macrophage activation to suppress the activity of infecting parasites. In an experimental murine leishmaniasis model, it was reported that the susceptibility of mice to L. major infection is due to the lack of Th1 responses related to IFN-y production and the development of Interleukin(IL)-4 dominated Th2 responses. However, despite of the importance of IFN-y in the resistance to L. major infection, several studies demonstrated that IFN-y alone is not sufficient to control L. major and that additional factor(s) would be required for the development of protective immunity to L. major. IFN-y is one of the macrophage-activating cytokine and activated macrophage is one of the principal source of IL-12. IL-12 is known as a cytokine involved in Th1 cell differentiation, and it stimulates NK cells to produce IFN-y. Moreover, IL-12 induces autocrine macrophage activation and consequently IL-12 and IFN-y production from the activated macrophages. Previously, it was reported the importance of IL-12 for resistance to L. major infection. It is therefore expected that both IFN-y and IL-12 are directly related to behavior of Leishmania in infected macrophages. Cytokines such as IFN-y enhanced nitric oxide (NO) production in macrophages by stimulating inducible nitric oxide synthase (iNOS) expression. NO plays an important role in clearance of various pathogens, and induction of NO production with IFN-y from macrophages has been demonstrated in several pathogen including Mycobacterium and Leishmnaia to control the growth of these intracellular pathogens. Therefore, it is expected that IFN-y and IL-12 are directly related to the behavior of Leishmania in infected macrophages.

In the second chapter, to investigate the effect of IFN-y and IL-12 on the susceptibility of macrophage to L. major infection, susceptible PMphi pretreated with IFN-g and/or IL-12 were infected with the parasites. Four hr p.i., PMphi treated with the combination of IFN-y and IL-12 (IFN-y/IL-12) showed significantly lower levels of the rate of infection and intracellular parasite than those of the nontreated cells. However, PMphi treated with either IFN-y or IL-12 did not show resistance to L. major infection. In addition, 72 hr p.i., the IFN-y/IL-12-treated and IFN-y-treated PMphi showed significantly lower levels of infection rate and intracelular parasite than those of the nontreated cells, and higher levels of resistance was observed in the IFN-y/IL-12-treated PMphi than in the IFN-y-treated PMphi. Although IFN-y/IL-12 treatment of PMphi prior to the infection led to the induction of resistance, as described above, this resistance was not induced when these cytokines and the parasites were added simultaneously to PMphi culture. These results suggest that IFN-y/IL-12 treatment prior to the infection restricts the early phase of the infection.

At the early point of infection, PMphi treated with IFN-y/IL-12 showed low levels of NO production and few L. major infection. On the other hand, nontreated control PMphi produced little NO at similar levels as cytokine-treated cells, and they showed higher infection rate and intracellular parasite compared to cytokine treated cells. Since the NO production of nontreated PMphi was comparable levels as cytokine treated cells, the susceptibility of untreated PMphi at the early phase of infection seemed to be due to mechanisms that were not associated with the parasiticidal NO activity. The amount of NO would be at base level so that it would not be associated to parasite elimination. At the late phase of infection, cytokine treated PMphi produced high levels of NO comparing to untreated cells, and this production might relate to the resistance to L. major infection in the cytokine-treated cells. Hence, the cytokine-induced resistance and NO production would correlate to lead PMphi to the resistance to L. major at this late phase. In the second chapter, it is demonstrated that treatment of IFN-y/IL-12 induce resistance to L. major, and the results indicated that the involvement of additional mechanisms in inducing resistance to L. major infection.

In the second chapter, it is showed that the combination of IFN-y and IL-12 induced the resistance to L. major at the early phase of infection. Since the event occurring at the early phase is the invasion of the parasite to macrophages, receptor molecules associated to the binding between parasite and macrophages were focused on next study. Macrophages have a pivotal role in both recognition and response to the infection at the early phase, primarily due to their ability to phagocytose. They eliminate pathogen, and affect the activity of other cells that critical in the immune response. Phagocytosis involves receptors on macrophages and it is known that the receptor expression levels are variable in each macrophage. The entry of L. major into macrophages is mediated by the recognition of specific parasite ligands by receptors. Complement receptor 3 (CR3) is highly expressed on a subset of macrophages. The surface of promastigotes is dominated by phosphoglycans (LPG) and the glycoprotein, gp63, and these molecules have been shown to be important in binding, invasion and intracellular survival of the parasites in macrophages. L. major promastigotes bind macrophages through these molecules mediated by CR3. Hence, CR3 would be strongly involved in parasite binding to macrophages in serum dependent and independent mechanism. It has been demonstrated that some cytokine affect receptor expression on macrophages, however, it is not known if IFN-y and IL-12 affect expression of the receptors that involved in parasite binding to macrophages and the resistance to the infection.

In the third chapter, to investigate association of the receptor expression levels and the susceptibility to L. major infection, CR3 expression on macrophages were examined under normal and cytokine treated condition. Surface expression of CR3 on AMphi and PMphi were examined using fluorescence activated cell sorting (FACS). PMphi showed high CR3 expressing phenotype, and as expected, quite low levels of CR3 expression was observed on AMphi. Next, to examine if the resistance of cytokine-treated macrophages associates to CR3 expression level, the expression of CR3 on macrophages were observed following their treatment with IFN-y and IL-12. Treatment with both IFN-y and IFN-y/IL-12 did not affect the expression of CR3 on PMphi comparing to nontreated PMphi. These results suggest that intracellular mechanisms, which regulate expression of other surface molecules, would be associated to inhibition of the parasite invasion that was induced by the cytokines. The result by flow cytometric analysis suggests the possibility that the different susceptibility to L. major infection in AMphi and PMphi would be related to the expression levels of CR3 on these macrophages. And the resistance of cytokine treated PMphi would associate to other factor derived from macrophages.

In this study, it is demonstrated that macrophages distributed in different site showed various susceptibility to L. major. Macrophages distributed in the lung where L. major infection is not documented, displayed resistance to the infection with low expression of CR3 on its surface. Susceptible macrophages resistance to L. major by the treatment of IFN-y/IL-12 prior to the infection, the resistance was not involved CR3 mediated mechanisms. Direct association of the receptor and the infection has not been demonstrated. However, the resistance in each macrophage would be mediated by different mechanisms.

The mechanisms of site-specific parasite infection remain poorly understood. In this study, it was found that macrophages distributed in different sites show different susceptibility to L. major infection, and AMphi were resistant comparing to PMphi. Also, susceptible PMphi induced resistance to the parasite by the treatment with IFN-y/IL12 prior to the infection. However, the observed resistance would involve different mechanisms. Further study on cytokine production and receptor expression in the tissues including the lung would provide better understanding of the factors critical for development of protective immunity and establishment of site-specific infection. And that will help to know the mechanisms of development of the susceptibility and the resistance of macrophages to Leishmania infection.

骨髄の造血幹細胞から血流に乗って様々な組織へ移動したマクロファージは組織マクロファージへと分化し、各組織に特色のある機能を担う。リーシュマニア症の原因となるリーシュマニア原虫は、マクロファージに感染し、分裂・増殖する。マクロファージは生体内に広く分布しているにもかかわらず、原虫感染はすべての組織で見られるわけではなく、また、同じ皮膚でも、原虫の侵入部位により異なる免疫応答が起こることが近年報告されているが、組織ごとの感染のメカニズムは已然不明である。本論文は、異なる組織のマクロファージの原虫感受性が異なるため、組織ごとに原虫感染の成否が異なるという仮説を基にマクロファージの原虫への感受性に関わる要因について解析し、リーシュマニア症発症における関与について考察しており、下記の以下の3章で構成されている。

第1章では、感染が見られない組織由来のマクロファージとして肺胞マクロファージを、また、原虫感染モデルとして広く用いられている腹腔マクロファージを原虫感受性のマクロファージをモデルとし、原虫感受性をin vitro原虫感染実験で調べたところ、感染のみられない肺由来のマクロファージと原虫感受性マクロファージとの原虫感受性が異なり、原虫が感染する組織と感染しない組織では、それぞれの組織に分布するマクロファージ自身の原虫感受性が違う可能性が示された。

第2章では、マクロファージの殺原虫活性を亢進するサイトカインであるインターフェロンy(IFN-y)およびインターロイキン12(IL-12)に着目し、原虫易感染性である腹腔マクロファージのリーシュマニア原虫感染性に対するIFN-yおよびIL-12の関与を検討した。マクロファージに、IFN-yとIL-12の一方であるいは同時に処理した後、原虫を感染させた。感染4時間後では、IFN-yとIL-12の両方(IFN-y/IL-12)で処理したマクロファージでは、未処理のものに比べ、原虫感染率および細胞内原虫数が有意に低下した。IFN-yまたはIL-12単独で処理したマクロファージはこれらの原虫抵抗性を示さなかった。感染72時間後には、IFN-y/IL-12あるいはIFN-yで処理したマクロファージにおいて、感染率および細胞内原虫数が未処理のものに比べ有意に低下したが、その効果は、IFN-y単独処理よりIFN-y/IL-12処理の方が高かった。これらの効果は、感染と同時にサイトカイン処理をしても認められなかった。以上の結果より、IFN-y/IL-12の前処理によってマクロファージは原虫感染初期に原虫抵抗性を獲得することが示唆された。また、感染後期の主要な殺原虫因子である一酸化窒素(NO)は、感染4時間後では、ほとんど産生されておらず、感染初期の原虫抵抗性にはNO以外の要因が原虫抵抗性を付与していることが示唆された。しかし、感染72時間後には、IFN-y/IL-12で処理したマクロファージで、高いNO産生が見られ、感染抵抗性との関連が示唆された。

第3章では、マクロファージの原虫感受性に感染初期に強く関与している要因として、マクロファージの原虫レセプターに着目し、原虫側リガンドとしてgp63やlipophosphoglycanが報告されている原虫レセプターのひとつである補体レセプター(CR3)発現量の(i)原虫感受性との関係、(ii)IFN-y/IL-12処理による変動の2点について解析した。(i) 抗CR3抗体を用いたフローサイトメトリーによる解析から、肺胞マクロファージではCR3の発現頻度が低く、腹腔マクロファージでは高く、両細胞間の原虫感受性の違いにCR3が関与している可能性が示された。(ii) IFN-y/IL-12処理により抵抗性を誘導された腹腔マクロファージにおいては、CR3の発現は減少しておらず、IFN-y/IL-12処理による抵抗性の誘導には、CR3以外のレセプターか、原虫接着後の感染プロセスにおけるマクロファージ活性化が関わっていると考えられた。

以上のように、本論文は、組織マクロファージによって原虫感受性が違うこと、易感染性マクロファージの感染感受性は、感染前のIFN-y/IL12への暴露により、感染初期に修飾されることを明らかにし、さらに、これらの感染初期の原虫感染抵抗性には、原虫レセプターを介した制御がありうることを提言しており、本論文の成果は、原虫の組織侵入・病変形成のメカニズムの解明および感染阻止免疫法の開発に有益な情報を提供しているもので、医学および獣医学の学術上貢献するところが少なくない。よって、審査員一同は、本論文が博士(農学)の学位論文として、価値あるものと認めた。