Role of Mononuclear Leukocytes in Immunity

描述(申请人提供)：我们已经使用三维纤维蛋白和I型胶原凝胶比较了组织状环境和搅拌悬浮液中中性粒细胞(PMN)的杀菌活性，发现需要一个临界PMN浓度(Cnc)来阻止纤维蛋白和胶原凝胶以及搅拌悬浮液中细菌的生长。执行特定免疫效应功能所需的临界白细胞浓度这一概念既新颖又重要。它统一了大量关于宿主防御细菌感染的文献，并提供了一个概念框架，用于定量评估必须交付给组织以执行特定功能的任何类别或类型的免疫细胞的浓度。我们已经推导出了一个方程式，使我们能够计算出目前测试的所有实验条件下所有细菌浓度的Cnc。利用它，我们发现悬浮液中的CnC(约4×105PMN/ml)与已知的使中性粒细胞减少的人易患脓毒症的CnC几乎相同，而纤维蛋白凝胶中的CnC则高出2.5-10倍(例如，1-4x106PMN/ml)。利用这些凝胶，我们扩展了之前的观察结果，即特定的基质蛋白(例如纤维蛋白)和趋化剂(例如fMLP)通过显示在纤维蛋白存在的情况下阻止PMN的杀菌活性来阻止PMN的迁移。此外，我们发现PMN在基因上缺乏a2-整合素的吞噬作用，在纤维蛋白凝胶中杀死表皮葡萄球菌的效率与野生型PMN一样高。我们还发现，与传统的看法相反，PMN有能力在成熟的(5天大的)生物膜中入侵和杀死98%的表皮葡萄球菌。我们寻求持续的支持，以扩大这些研究，并确定调节PMN和单核细胞从血液向组织迁移的机制，限制组织损伤的关键过程，以及提供足够的PMN和单核细胞以消除浮游细菌和生物被膜细菌，以及细胞毒淋巴细胞以杀死癌细胞。这个应用程序有三个具体目标。目的：1.测定单核细胞在纤维蛋白和I型胶原凝胶中体外杀灭表皮葡萄球菌和大肠杆菌的临界浓度，以及体内杀灭表皮葡萄球菌和大肠杆菌的单核细胞的临界浓度，以确定PMN和单核细胞停止进入细菌感染的真皮部位的机制。目的#2.确定阻止中性粒细胞和单核细胞杀伤生物材料相关的表皮葡萄球菌生物膜的免疫学机制。目的#3.确定临界白细胞浓度的概念是否也适用于细胞毒性淋巴细胞和单核细胞的杀瘤活性。换句话说，细胞毒性淋巴细胞必须达到肿瘤床内的临界浓度才能减少肿瘤质量吗？

DESCRIPTION (provided by applicant): We have used three-dimensional fibrin and collagen I gels to compare neutrophil (PMN) bactericidal activity in tissue-like environments vs. stirred suspensions and discovered that a critical PMN concentration (CNC) is required to block the growth of bacteria in fibrin and collagen gels and in stirred suspensions. The concept that a critical leukocyte concentration is required to carry out specific immune effector functions is both novel and important. It unifies a large body of literature on host defense against bacterial infections, and provides a conceptual framework for assessing quantitatively the concentration of any class or type of immune cell that must be delivered to a tissue to execute a specific function. We have derived an equation that enables us to calculate the CNC for all bacterial concentrations under all experimental conditions so far tested. Using it we found the CNC in suspension (approximately 4 x 105 PMN/ml), is almost identical to that known to predispose neutropenic humans to sepsis, while that in fibrin gels is 2.5 - 10-fold higher (e.g., 1-4 x 106PMN/ml). Using these gels we have extended our previous observation that specific matrix proteins (e.g., fibrin) and chemoattractants (e.g., fMLP) block PMN migration by showing that in the presence of fibrin, fMLP blocks PMN bactericidal activity. Moreover, we discovered that PMN genetically deficient in a2-integrins phagocytose and kill S.epidermidis as efficiently as wild-type PMN in fibrin gels. We also have discovered that contrary to conventional wisdom, PMN have the capacity to invade and kill >98% of S. epidermidis in mature (5-day old) biofilms. We seek continued support to extend these studies, and to identify mechanisms that regulate emigration of PMN and monocytes from the blood into tissues, processes central to limiting tissue damage, and to delivering sufficient PMN and monocytes to eradicate planktonic and biofilm bacteria, and cytotoxic lymphocytes to kill cancer cells. This application has three Specific Aims. Aim #1. To measure the critical concentration of monocytes for killing S. epidermidis and E. coli in fibrin and collagen I gels in vitro, and the critical concentrations of PMN and of monocytes for killing of S. epidermidis and E.coli in vivo, and to identify the mechanisms that signal cessation of entry of PMN and of monocytes into dermal sites of bacterial infection. Aim #2. To identify the immunological mechanisms that impede PMN and monocyte killing of biomaterials-associated S. epidermidis biofilms. Aim #3. To determine whether the concept of a critical leukocyte concentration also applies to the tumoricidal activities of cytotoxic lymphocytes and monocytes. In other words, must cytotoxic lymphocytes reach a critical concentration within a tumor bed to effect a reduction in tumor mass?