Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3Kδ in neutrophil chemotaxis, we have developed a potent and selective PI3Kδ inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3Kδ activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3Kδ, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3Kδ can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.

Human bone marrow-derived CD34+ cells were analyzed for the expression of the beta 1-family of integrin adhesion molecules. Integrin alpha 4 beta 1 was consistently expressed by greater than 90% of CD34+ cells, including essentially all assayable granulocyte-macrophage colony- forming cells (CFU-GM) and erythroid bursts (BFU-E) as shown by fluorescence-activated cell sorting studies. Adhesion of highly enriched CD34+ cells to cultured allogeneic marrow stromal cells was largely inhibited both by monoclonal antibody to alpha 4 beta 1 and to vascular cell adhesion molecule-1 (VCAM-1), a ligand for alpha 4 beta 1. VCAM-1 was found to be expressed by bone marrow stromal elements in vitro both constitutively at low level and at high levels after treatment with cytokines. Induction of VCAM-1 was cytokine- and time- dependent with maximum levels being obtained after 4 hours of exposure to a combination of interleukin-4 and tumor necrosis factor-alpha. Cytokine-induced stromal cells bound threefold higher numbers of CFU-GM and BFU-E, this increase being abrogated by anti-alpha 4 beta 1 and anti-VCAM-1 antibodies. In addition, the adhesion to stroma of more immature progenitors, the long-term culture initiating cells, also occurred through an alpha 4 beta 1/VCAM-1-dependent mechanism. These studies identify an adhesion mechanism of potential importance in the localization of primitive progenitors within the hematopoietic microenvironment.

We reported a development of murine monoclonal antibodies to a molluscan small cardioactive peptide (SCPB) and their application to immunolabeling of neurons in several molluscan and arthropod species. In vitro stimulations of mouse lymphocytes with SCPB conjugated to a carrier protein yielded exclusively IgM class antibodies; in vivo stimulation resulted in generation of both IgM and IgG classes of antibodies. Monoclonal antibodies of the IgM class labeled identified SCP-containing neuron B11 in the frozen sections of the buccal ganglia of Tritonia diomedia. These antibodies failed to stain any neurons in whole mount preparations. A monoclonal antibody of IgG1 subclass selectively labeled neurons in both frozen sections and whole mount preparations of diverse invertebrate species. Thus, neurons B11, B12, and GE1 and several other neurons of the buccal and gastroesophageal ganglia of T. diomedia bound the antibody, and a similar pattern of immunolabeling was found in the closely related gastropod Tritonia festiva. We also observed SCPB-like immunoreactivity in the central neurons of other nudibranch and pulmonate molluscs and in examples of insect (Acheta domesticus and Tehrmobia domestica) and crustacean (Semibalanus cariosus) classes of the Arthropoda. Our results suggest a specific pattern of distribution of SCPB-like immunoreactivity in the gastropod nervous system and a broad occurrence of SCPB-like antigenicity in the diverse invertebrates.

SCP-like antigenicity is first present in Tritonia diomedea in small cells of the cerebral ganglia and a single axon crossing the cerebral commissure of 8-day-old embryos. Other axons and neurons become antigenic as the larva develops. At 4-9 days after larvae hatch from the egg mass, 2 additional pairs of neurons are labeled. Axons extend from one pair to the left cerebral ganglion and from the other to the right. A second labeled axon is present across the cerebral commissure. In metamorphically competent larvae the cerebral and pedal neuropils, as well as two neurons in the buccal ganglia with axon(s?) across the commissure, are antigenic. The change in antigenicity as the larva becomes competent is presumably preparatory for juvenile life. The labeled buccal neurons may be B12, which are known to contain SCPs, extend an axon across the buccal commissure, and function in adult feeding behavior. The two large neurons strongly labeled by rabbit polyclonal antibodies against FMRFamide are clearly different from neurons labeled by monoclonal antibody against SCPs. This result supports the contention that different antigens are labeled by these two immune probes.

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