Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry.

In order to better understand the role of lysophosphatidic acid (LPA) in physiology and pathophysiology, it is necessary to accurately determine the molecular species and amounts of LPA in biological samples. We have developed a stable-isotope dilution, liquid chromatography-mass spectrometry assay for the direct quantitative analysis of 1-acyl-LPA. This method utilizes a deuterium-labeled internal standard, LPA (18:0-d(35)), and a single liquid-liquid extraction with acidic butanol that allows >95% recovery of LPA, followed by online normal-phase liquid chromatography-mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the individual 1-acyl-LPA species present in biological samples. The utility of the assay is demonstrated through the analysis of LPA species in plasma and serum from human volunteers. Total LPA in EDTA plasma was 0.61 +/- 0.14 microM in males and 0.74 +/- 0.17 microM in females, which increased to 0.91 +/- 0.23 and 0.99 +/- 0.38 microM after incubation for 24 h at 25 degrees C. Total LPA in serum was 0.85 +/- 0.22 microM in males and 1.57 +/- 0.56 microM in females, which increased to 4.78 +/- 0.89 and 5.57 +/- 0.73 microM after incubation for 24 h at 25 degrees C.

Phospholipid growth factors and corneal wound healing.

In many tissue types, wound healing involves cell division and migration over and into the wound area to cover and remodel the wound. LPA and other members of the phospholipid lipid growth factor (PLGF) family stimulate many of the activities involved in wound healing. In the rabbit cornea, we have found that keratocytes from wounded corneas have a volume-activated Cl- current activated by LPA and alkenyl-LPA. This current is minimally activated by cyclic PA and SPC, and is not activated by LPA in cells from uninjured corneas. Biochemical examination of PLGFs in aqueous humor and lacrimal fluid before and after wounding identified LPA, alkenyl-GP, PA, and lyso PS, with elevated PLGF activity after wounding. In recent experiments examining human corneal cell lines and cultured cells using RT-PCR, we found mRNA for EDG receptors 1-5, with an apparent increase in EDG-3, -4, and -5 following brief SDS application to cell lines, and EDG receptors 2-5 induction in late-passage human corneal epithelial cells. This work points to a significant role for PLGFs in the corneal wound-healing process.

Role of lysophosphatidic acid in endothelin-1- and hematoma-induced alteration of cerebral microcirculation.

Cerebral hematoma increases cerebrospinal fluid (CSF) endothelin-1 (ET-1). Inhibitors of ET-1 synthesis prevent this increment and hematoma-induced modification of cerebral arteriolar reactivity. We hypothesized that intrathecal ET-1 injection could 1) modify pial arteriolar reactivity similarly to hematoma; 2) increase CSF lysophosphatidic acid (LPA), a potential contributor to altered cerebrovascular reactivity; and 3) reduce the level of adenosine 3',5'-cyclic monophosphate (cAMP) in the CSF. Either ET-1 (10(-7) M) or artificial CSF was injected over the left parietal cortex of newborn pigs. Four days later, cranial windows were implanted. CSF ET was increased from a basal level of 11 fmol/ml to 18 fmol/ml 4 days after ET-1 injection, whereas CSF cAMP was reduced from 2,700 to 950 fmol/ml. The mean diameter of pial arterioles was reduced 31%. In control animals, 10(-12) M ET caused dilation, and higher concentrations induced vasoconstriction. Four days after ET-1 injection topical ET-1 caused constriction instead of dilation at 10(-12) M, and constrictions at higher doses were enhanced. Norepinephrine-induced constrictions were potentiated in the ET-1-injected group. Dilations to cAMP-dependent (but not independent) vasodilators were attenuated after ET-1. The concentration of the vasoconstrictor lipid mediator LPA increased approximately fourfold. Thus intrathecal injection of ET-1 mimics hematoma-induced modification of cerebral vascular reactivity and increase in LPA production. The mechanism(s) of ET-1- and hematoma-induced modifications may involve LPA, which is known to contribute to the loss of dilator responses by inhibition of cAMP product on. The present study further suggests that ET-1 together with LPA could be causing changes in cerebrovascular reactivity following cerebral hemorrhage. ET-1 stimulates the release of LPA from brain parenchyma independent of serum so that LPA could serve as a secondary mediator.

Xenopus oocytes as immunological vectors to produce monoclonal antibodies to rat brain antigens.

A novel approach was developed to raise a panel of monoclonal antibodies (mAb) against brain antigens using Xenopus oocytes as immunological vectors. Xenopus oocytes were injected to express proteins encoded by brain-derived mRNA extracted from rat cerebral cortex. A crude membrane preparation from mRNA-injected oocytes was then used to immunize mice previously rendered immunotolerant to native oocyte membranes. mAb reacting with cryostat cut sections from rat brain were selected and further characterized by immunohistological and immunobiochemical techniques. Several mAb recognized brain specific antigens, including some that were cell type specific and others that revealed a regional binding pattern. A particular group of antibodies recognized an epitope localized exclusively to the cerebellar pinceau terminals. Although some of the hybridomas found in this panel may be products of natural autoreactive lymphocytes, the presence of a specific immune response to mRNA expression products is discussed. These results indicate that mRNA injected oocytes are useful tools to raise mAb to study the molecular diversity of the nervous system.

Modulation of cell-cell and cell-antigen interactions by 1,25-dihydroxyvitamin D3 and vitamin D3 sulfate in vitro: a study on pregnancy lymphocytes and hybridoma cells.

The effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was investigated in single cell cytotoxicity assays, using K-562 target cells. The action of vitamin D3 sulfate (VD3S) in natural cytotoxicity assays as well as its effect on the antigen-specific adherence of hybridoma cells has also been studied. In the single cell cytotoxicity assay 1,25(OH)2D3 dose-dependently and significantly increased the binding of PBMC to target, the number of lysed target cells and NK activity. RU486, a compound known as a potent blocker of progesterone and glucocorticoid receptors, suppressed the effect of 1,25(OH)2D3 in all systems. VD3S dose-dependently decreased the natural cytotoxicity of PBMC and the binding of hybridoma cells to antigen immobilized on plastic surfaces. The results suggest that both 1,25(OH)2D3 and VD3S are potent modulatory agents in cell-cell and cell-antigen interactions.

Adherence of cells to myelin basic protein. I. Adherence of red and white blood cells from patients with multiple sclerosis to myelin basic protein.

Red blood cells (RBC) and white blood cells (WBC) of patients with multiple sclerosis (MS) show decreased adherence to myelin basic protein (MBP) immobilized on plastic surfaces compared to the binding of cells from patients with other neurological diseases (OND), or such other autoimmune diseases as psoriasis (PS), and to that of healthy controls (HC). No similar phenomenon occurred to basic and non-basic type proteins other than MBP, for example, to histone (HIS), lysozyme (LYS) and ovalbumin (OVA). Thus, decreased adherence of RBC and WBC in MS patients to MBP appears to be a unique feature of the disease if compared with OND or PS.

Adherence of cells to myelin basic protein. II. Adherence of red blood cells of SJL mice with chronic relapsing EAE.

Adherence of red blood cells from SJL mice suffering of chronic relapsing experimental allergic encephalomyelitis was studied to myelin basic protein coated microtiter plates. Control animals received either bovine serum albumine or "protein-antigen free" adjuvant using the same immunization protocol. Characteristic changes in adherence were found in bovine or human myelin basic protein injected animals compared to the bovine serum albumine immunized group. After a nonspecific increase in adherence between Days 2 to 6 observed in all 3 groups, in the encephalitogen challenged animals on Days 13-14 a marked decrease in red blood cell adherence was detected which maintained at this decreased level during the clinically active stage of the disease and reappeared with the relapse of EAE. No such decreased adherence of red blood cells was observed in BSA immunized animals or in adherence of cells from myelin basic protein injected animals to other basic type protein such as histone. Thus, decreased adherence of red blood cells in animals with EAE appears to be an interestingly unique measure of the disease activity.

Antigen-specific cell adherence assay: a new method for separation of antigen-specific hybridoma cells.

A new method for the detection and separation of antigen-specific antibody-producing cells on the basis of antibody-mediated recognition of solid-phase immobilized antigen molecules is described. Hybridoma cells are placed on microtiter plate wells coated with antigen molecules, and antigen-specific antibody-producing cells bind to the immobilized antigen molecules; antibody nonproducing or nonspecific antibody-producing cells can be easily separated from the bound cells by inverting the plate. Cells bound to solid-phase immobilized antigen molecules can readily be quantitated by counting under a light microscope, and the cells recovered can produce antibody in culture. Unspecific binding of cells in antigen-specific cell adherence assay (ASCAA) is optimally below 5%. Also, effect of drugs interfering with processes related to antibody production of antigen-specific cells can be detected and evaluated by ASCAA.

Antigen-specific adherence of antibody-producing hybridoma cells: application to neural and nonneural antigens.

Antibody-producing hybridoma cells specifically bind to microgram quantities of antigen molecules adsorbed onto the surface of plastic microtiter plates. The binding of hybridoma cells to nonantigen is optimally below 5%, similar binding of non-antibody-producing cells is 4-7%, compared to the binding of the hybridomas to their antigen. There is a difference in the kinetics of binding hybridomas to antigen compared to nonantigen. The number of bound cells depends on the amount, i.e., the surface density, of the antigen molecules and shows typical saturation effects. Preincubation of hybridomas with excess free antigen and saturation of the antibody binding site on the surface with the hybridoma-produced antibody reduces binding of the hybridoma cells to the antigen. Treatment of cells with trypsin reduces binding to antigen-coated plastic surfaces. Drugs such as sodium azide, cytochalasin B, colchicine, vinpocetine, and vincristine sulfate reduce binding to the antigen. Hybridoma cells adhering to the antigen produce more antibody than nonadhering cells. The results reported in this paper show that antigen molecules adsorbed to include a plastic surface and hybridoma cells interact specifically. This system forms a suitable model to study the interaction of antigen with antigen-specific cells and may be useful as a separation method for specific antibody-producing cells.

Isolation of the human myelin basic protein by immunoaffinity chromatography with a monoclonal antibody.

Immunoaffinity chromatography has been developed for the isolation of the human myelin basic protein (MBP). The method is based on the use of a monoclonal antibody which was produced to bovine MBP, cross-reacting with human MBP. The protein isolated from acidic extracts of the brain proteins was shown to be native MBP by its immunochemical reactivity, by its ability to elicit experimental allergic encephalomyelitis and by its mol. wt (18,600 +/- 400). It represented a single-band purity after hypersensitive silver staining. The MBP isolated by the method described represents a higher purity than that of the MBP purified by conventional multistep biochemical separation techniques.