In vivo administration of interleukin 1 elicits increased Ia antigen expression on B cells through the induction of interleukin 4

The effects of the in vivo administration of interleukin 1 (IL 1) on lymphocytes from lymph node and spleen were analyzed. Mice received five daily subcutaneous (s.c.) injections of various doses of human recombinant IL 1 beta. Either 1 or 7 days after IL 1 treatment, spleens, popliteal and inguinal lymph nodes were collected. Lymphadenosis and splenomegaly were observed in the IL 1-treated animals. Lymph nodes from IL 1-treated mice contained a higher percentage of B cells than controls, and B cells from IL 1-treated mice expressed dramatically increased levels of Ia antigen. Lymphadenosis and splenomegaly, as well as the changes in subset distributions and Ia expression were transient. Concomitant treatment of mice with IL 1 and anti-IL 4 monoclonal antibody suppressed IL 1 effects on B cell Ia expression, but not on the B/T cell ratio. In situ hybridization analyses revealed that IL 1 treatment induced the expression of mRNA for IL 4, interferon-gamma, and IL 2 in lymph node and spleen cells. The distribution of cells expressing the various cytokine mRNA was markedly different between the spleens and lymph nodes.     

In vivo induction of hepatic p-glycoprotein by cyclosporine in the rat.

The objective of the present study was to investigate the regulation of P-glycoprotein by cyclosporine, a known inhibitor of CYP3A, at different dosage levels and lengths of treatment. Rats were given various doses of cyclosporine through oral administration or subcutaneous injection. Each treatment group was studied for 28 days or 28 days followed by 14 days of olive oil vehicle dosing. In each group, rats administered vehicle alone served as the controls. At the end of the study, liver microsomes were prepared and hepatic P-glycoprotein levels were quantified by Western blot analysis. Significant induction of hepatic P-glycoprotein was found in rats given cyclosporine. Rats administered 30 mg/Kg/d orally and 15 mg/Kg/d subcutaneously showed an increase in hepatic P-glycoprotein by 93% (p = 0.0011) and 136% (p < 0.001), respectively. Low doses of cyclosporine also induced P-glycoprotein but not to a significant extent, indicating a dose-dependent effect. The pattern of induction of P-glycoprotein was not, however, dependent on the route of administration. Fourteen days after the discontinuation of cyclosporine treatment, P-glycoprotein levels returned to near the control values. As a drug efflux transporter, the induction of P-glycoprotein by cyclosporine may decrease the hepatic metabolism of P-glycoprotein substrates. Therefore this induction of hepatic P-glycoprotein and suppression of hepatic CYP3A may have a coordinate effect on the metabolism of cyclosporine. These data may help explain the large variability associated with cyclosporine absorption, metabolism, and circulating blood levels.     

In vivo labeling of resident peritoneal macrophages

A novel method for labeling resident peritoneal macrophages (M phi) by injection of a dye into the peritoneal cavity is described. The dye, which fluoresces green, is selectively taken up by the resident M phi. Dye labeled cells can be further characterized by labeling of cell surface antigens with monoclonal antibodies (Mabs) and phycoerythrin conjugated second antibody. After such labeling with the Mabs F4/80 or Mac 1 the resident M phi were labeled by both the green dye and the red Mab markers, while recruited M phi or neutrophils were labeled with just the red Mab; the two populations of cells were readily distinguished by two-color flow cytometry. This technique enabled identification of resident and recruited M phi in each animal without the use of radioisotopes, irradiation, or bone marrow ablation. Sufficient numbers of cells can be analyzed from each animal so that individual animals could be evaluated. We found no adverse effects of this labeling technique on expression of cell surface antigens or M phi mediated cytotoxicity. We did find evidence that the i.p. injection induced a mild inflammation in the peritoneal cavities of animals injected with either the dye or the balanced salt solution vehicle. Examination of the intracellular staining pattern indicated that the label rapidly sequestered in the cytoplasm of the M phi, possibly in the lysosomes. Dye solubility studies showed that the dye was partially soluble at the concentration used for in vivo labeling. We hypothesize that the M phi labeling occurred by a combination of phagocytosis of dye aggregates and endocytosis of labeled plasma membrane.     

Pharmacological modulation of rat monocytes:In vivo effects on Ia expression and interleukin-1 production

We have shown that during the developing phase of adjuvant disease (AD) in rats the expression of MHC class II (Ia) antigens on blood monocytes (BM) was enhanced. The results of a study in established AD are reported now. Four agents were tested: indomethacin and diclofenac-sodium (1 mg/kg/day); levamisole and prinomide (10 mg/kg/day), administered orally from day 18–31 after induction of AD. We assessed the following BM parameters: Ia expression, interleukin-1 (sIL-1) production, and membrane bound IL-1 (mIL-1). In AD Ia expression was enhanced, no changes occurred in mIL-1 or sIL-1. Indomethacin treatment reduced sIL-1 production, levamisole Ia expression and mIL-1 activity, prinomide all three parameters measured and diclofenac, though clinically effective, none.     

Scanning electron microscopic study of the renal glomerulus by an in vivo cryotechnique combined with freeze-substitution

The 3-dimensional ultrastructure of mouse renal glomeruli under normal haemodynamic conditions was studied by scanning electron microscopy using an in vivo cryotechnique followed by freeze-substitution, and compared with glomeruli prepared by conventional fixation methods. Mouse kidneys were frozen with a cryoknife apparatus and a liquid isopentane-propane mixture (−193°C). Surface areas of the frozen tissues were freeze-fractured with a scalpel in liquid nitrogen. The specimens were routinely freeze-substituted, freeze-dried, ion-sputtered, and then observed in a scanning electron microscope at an accelerating voltage of 5 kV. Renal glomeruli showed good ultrastructural preservation of the surface tissues. Podocytes with interdigitating foot processes covering capillary loops exhibited smooth surface contours and their cell surfaces were arranged more tightly than those seen by the conventional fixation method. Filtration slits between foot processes were found to be narrow. The internal structure of the glomerular tuft was seen in the freeze-fracture faces. The capillary lumen with variously shaped erythrocytes was kept open in frozen glomeruli under normal blood circulation conditions. The ultrastructure of renal glomeruli, as revealed by the in vivo cryotechnique with freeze-substitution, appears to be closer to that of the living state.     

In vivo regulation of the Vi antigen in Salmonella and induction of immune responses with an in vivo-inducible promoter

Salmonella enterica serovar Typhi, the agent of typhoid fever in humans, expresses the surface Vi polysaccharide antigen that contributes to virulence. However, Vi expression can also be detrimental to some key steps of S. Typhi infectivity, for example, invasion, and Vi is the target of protective immune responses. We used a strain of S. Typhimurium carrying the whole Salmonella pathogenicity island 7 (SPI-7) to monitor in vivo Vi expression within phagocytic cells of mice at different times after systemic infection. We also tested whether it is possible to modulate Vi expression via the use of in vivo-inducible promoters and whether this would trigger anti-Vi antibodies through the use of Vi-expressing live bacteria. Our results show that Vi expression in the liver and spleen is downregulated with the progression of infection and that the Vi-negative population of bacteria becomes prevalent by day 4 postinfection. Furthermore, we showed that replacing the natural tviA promoter with the promoter of the SPI-2 gene ssaG resulted in sustained Vi expression in the tissues. Intravenous or oral infection of mice with a strain of S. Typhimurium expressing Vi under the control of the ssaG promoter triggered detectable levels of all IgG subclasses specific for Vi. Our work highlights that Vi is downregulated in vivo and provides proof of principle that it is possible to generate a live attenuated vaccine that induces Vi-specific antibodies after single oral administration.     

Involvement of inflammatory leukocytes in hepatic induction of T-kininogen in rat

Hepatic synthesis as well as plasma levels of T-kininogen, a protein precursor of T-kinin (Ile-Ser-bradykinin), increase in rats following the induction of acute inflammation. Studies have been undertaken to elucidate an involvement of inflammatory leukocytes in the acute-phase response of T-kininogen. Peritoneal exudate cells (PEC) were prepared from rats six days after the intraperitoneal injection of Freund's complete adjuvant. By transfer of these leukocytes into the peritoneal cavity of normal rats, plasma kininogen levels of these recipients increased about threefold after one day. Secretion of kininogen from rat hepatocytes in primary culture was enhanced about twofold by coculturing with PEC. A similar effect was also obtained by adding culture supernatant of these leukocytes into hepatocytes, and the increased levels of kininogen in culture medium of hepatocytes was due to the increased levels of T-kininogen. From these results, it was concluded that leukocytes in the inflammatory site, probably macrophages, release some substance(s) which stimulate(s) the hepatic synthesis of T-kininogen.This study was supported in part by a Grant-in-Aid from the Naito Foundation and one (No. 61580154) from the Ministry of Education, Science and Culture of Japan.     

Adenine uptake cells in the rat kidney with remarks on the expression of Ia antigen

The patterns of [14C] adenine ([14C]A) incorporation into DNA by proliferative cells in the kidney were studied by the autoradiographic technique. It was revealed that, after 3 daily injections of [14C]A (1 microCi/g body weight each), a portion of the glomerular cells and a few fibroblastoid cells in the cortical intexstitium incorporated [14C]A into DNA to a remarkable extent. Such cells also incorporated [3H]thymidine, but to a lesser extent. The cells which incorporate [14C]A to a particularly great extent (adenine uptake cells) also occur in other tissues. Such cells are confined to a few cell types of either the macrophage or fibroblast or reticulum cell lines. This fact suggests that the adenine uptake cells observed in the glomerulus are also of a similar cell line and most likely mesangial cells. By immunohistochemical examination for Ia antigen, adenine uptake cells are divided into Ia-positive and Ia-negative types. The present examination showed that the major portion of adenine uptake cells in the glomerulus are Ia-negative, and it is suggested that these cells are analogous to the Ia-negative macrophages in the lung. This suggestion is supported by the fact that, in the glomerulus, colloidal carbon uptake cells (macrophage-like cells) are present in fairly large numbers. The Ia-positive cells seem to be of the same cell line as the adenine uptake cells that express Ia antigen in other tissues, such as septal fibroblasts in the lung.     

Role of Ia antigen in the induction of adoptively transferred acute and chronic relapsing demyelinating disease in mice

Acute and chronic relapsing forms of experimental allergic encephalomyelitis (EAE) can be induced in SJL/J mice following transfer of myelin basic protein (MBP)-sensitized T cells which have been challenged in vitro with MBP. In this study, addition of specific anti I-A antibody during the culture blocked the antigen-specific proliferation of T cells and inhibited the transfer of both acute and relapsing EAE. Treatment of T cell recipients with anti I-As antibody daily for 10 days suppressed the induction of acute EAE. Further treatment of mice with anti I-As antibody reduced the number of relapses and improved their conditions. We conclude that MBP-sensitized T cells interact with Ia positive cells, both in vitro and in vivo, to induce acute and chronic relapsing EAE, respectively. The mechanism of this interaction and its role in the disease process are discussed.     

Surface charge distribution is a determinant of antigen deposition in the renal glomerulus: studies employing ''charge-hybrid'' molecules.

The deposition of antigens and immune complexes (IC) in the renal glomerulus is charge-dependent. The demonstration that molecules of net anionic charge, but with discrete positively charged regions, exhibit affinity for the glomerular basement membrane (GBM) extends this concept. Charge hybrid (polar) molecules were constructed by covalently coupling small polycations (lysozyme or linear poly-L-lysine chains with a mean of 17 and 20 residues) to larger polyanions (ovalbumin or human serum albumin (HSA]. Although the products were of overall net anionic charge they still bound to glomerular structures. Immunofluorescence studies performed after i.v. injection of the samples into rats revealed that HSA:poly-L-lysine had the highest affinity. Radioisotopic measurements showed uptake of HSA:poly-L-lysine to be a function of the number of lysine residues; binding of HSA:poly-L-lysine20 was 2.5 times higher than HSA:poly-L-lysine17 (P less than 0.01). Prior injection of a small competing polycation (polyethyleneimine 1200) reduced uptake of HSA:poly-L-lysine by 75%, indicating the charge-based nature of the interaction. HSA:poly-L-lysine20 alone was effectively eliminated from the glomeruli within 72 h. Administration of HSA:poly-L-lysine followed by anti-HSA antibody induced immune complex formation in the capillary wall, giving rise to a granular immunofluorescence pattern and discrete subendothelial and subepithelial deposits. Molecules with polar structure do occur naturally and may contribute to immune complex formation in glomerulonephritis.     

Immunoelectron microscopic analysis of Ia antigen expression in rat skin during limb allograft rejection

Using a whole-limb graft model in rats, morphologic changes and variations in the expression of Ia antigen on epidermal cells were investigated in the allografted skin during acute rejection. BN right limbs were transplanted to F344 recipients. Skin tissues were excised during acute rejection on days 1, 3, 5, 7, and 9 after the transplantation. Sections were examined for Ia antigen expression using immunohistologic techniques, and in situ quantification of Ia antigen was made using an immunogold method. Epidermal keratinocytes expressed Ia antigen before the grafts were rejected and the amount of Ia antigen expression increased and exceeded the amount of Ia antigen of Langerhans cells during the course of rejection. The progressive increase in class II antigen expression on EKs correlated with the appearance and relative accumulation of dermal lymphocytic cells. On the other hand, Ia antigen was not expressed on vascular endothelial cells during rejection. Our results suggest that the Ia-positive keratinocytes can serve as target cells in skin rejection of limb allografts. The immunogold technique we used seems most pertinent for a quantitative examination of cell-surface antigens in situ.     

In vivo viscoelastic properties of the brain in normal pressure hydrocephalus

Nearly half a century after the first report of normal pressure hydrocephalus (NPH), the pathophysiological cause of the disease still remains unclear. Several theories about the cause and development of NPH emphasize disease-related alterations of the mechanical properties of the brain. MR elastography (MRE) uniquely allows the measurement of viscoelastic constants of the living brain without intervention. In this study, 20 patients (mean age, 69.1 years; nine men, 11 women) with idiopathic (n = 15) and secondary (n = 5) NPH were examined by cerebral multifrequency MRE and compared with 25 healthy volunteers (mean age, 62.1 years; 10 men, 15 women). Viscoelastic constants related to the stiffness (μ) and micromechanical connectivity (α) of brain tissue were derived from the dynamics of storage and loss moduli within the experimentally achieved frequency range of 25-62.5 Hz. In patients with NPH, both storage and loss moduli decreased, corresponding to a softening of brain tissue of about 20% compared with healthy volunteers (p < 0.001). This loss of rigidity was accompanied by a decreasing α parameter (9%, p < 0.001), indicating an alteration in the microstructural connectivity of brain tissue during NPH. This disease-related decrease in viscoelastic constants was even more pronounced in the periventricular region of the brain. The results demonstrate distinct tissue degradation associated with NPH. Further studies are required to investigate the source of mechanical tissue damage as a potential cause of NPH-related ventricular expansions and clinical symptoms.