The cytokines NAP-1 (IL-8), MCP-1, IL-1 beta,and GRO in rabbit inflammatory skin lesions produced by the chemical irritant sulfur mustard

Developing and healing dermal inflammatory lesions were produced in rabbits by the topical application of dilute sulfur mustard (SM),⁹ the military vesicant. In tissue sections of such lesions, cells containing the mRNA of important cytokines were identified with in situ hydridization techniques. These cytokines were neutrophil attractant/activation protein-1 (NAP-1 (also called IL-8)), monocyte chemoattractant (activating) protein 1 (MCP-1), interleukin 1 (beta) (IL-1 (beta)), and GRO (a growth factor and chemokine). Mononuclear cells (mainly macrophages and activated fibroblasts) contained the mRNA of all four of these cytokines. A higher percentage of cytokine-producing mononuclear cells (macrophages and activated fibroblasts) was present in lesions at 2 days (their peak size) than at 6 days, when they were almost healed. Granulocytes emigrated from the bloodstream, passed through the lesions, and were the major constituent of the protective crust. This sequence correlated with the distribution of cells able to produce NAP-1: At 2 days and 6 days, the mononuclears that contained messenger RNA for this granulocyte chemoattractant were found mainly in the upper part of the dermis. At 2 days and 6 days, cells containing the mRNA of IL-1, a primary cytokine, were also found predominantly in the upper dermis, i.e., nearest the site of injury. In contrast, mononuclears containing the mRNA of MCP-1 (a monocyte chemoattractant), and the mRNA of GRO (a granulocyte chemoattractant) were more equally distributed throughout the dermis. SM stimulated hair follicle epithelial cells to up-regulate GRO mRNA and, to a lesser degree, NAP-1 mRNA. Apparently, the irritation produced by SM directly or indirectly induces such epithelial cells to manufacture these growth factors. In the rabbit, hair follicles are known to be the main source of new epithelial cells after the covering epithelium has been destroyed. Therefore, GRO is probably a major autocrine-paracrine stimulus for such repair. A brief review of the role of cytokines in dermal inflammation is presented.Abbreviations SM Sulfur mustard: bis(2-chloroethyl)sulfide - GM-CSF Granulocyte-Macrophage Colony Stimulating Factor - GRO A member of the CXC subfamily of chemokines that promotes the multiplication of cells, formerly called melanoma growth stimulating activity (MGSA) - IFN (gamma)-Interferon-gamma - IL-1 Interleukin 1 - IL-8 Interleukin 8 (same as NAP-1)-a CXC chemokine - MCP-1 Monocy te Chemoattractant (Activating) Protein-1-a C-C chemokine - NAP-1 Neutrophil Attractant/Activating Protein-1 (same as IL-8)-a C-X-C chemokine - TGF (beta) Transforming Growth Factor (beta) - TNF (alpha) Tumor Necrosis Factor (alpha) - EDTA Ethylenediamine tetraacetate - DEPC Diethylpyrocarbonate - PBS Phosphate-buffered saline solution - PGE₂ Prostaglandin E₂ - PGI₂ Prostaglandin I₂ (prostacyclin) - SSC Sodium chloride-sodium citrate solution On leave of absence from the Institute for Medical Immunology, Kumamoto University School of Medicine, Kumamoto, Japan.On leave of absence from the Department of Internal Medicine, Oita Medical University, Oita, Japan.     

An improved technique for repeated gavage administration to rat neonates

ABSTRACT  The technique for gavage administration to rat nurslings was improved to allow determination of the direct effects of chemical substances in the nurslings. Rat neonates were treated with distilled water from postnatal day 1 through 20 using this technique. The viability of neonates during the administration period was comparable to that of untreated neonates. No adverse effects of this technique on the development of neonates were found, and no histological alterations of the esophagus or pharynx. Therefore, we conclude that use of our improved gavage administration method will contribute to ensuring successful neonatal development and thus allowing accurate assessment of the toxicological effects of test compounds on rat nurslings.     

Cell-type-specific excitatory and inhibitory circuits involving primary afferents in the substantia gelatinosa of the rat spinal dorsal horn in vitro

The substantia gelatinosa (SG) of the spinal dorsal horn shows significant morphological heterogeneity and receives primary afferent input predominantly from Aδ- and C-fibres. Despite numerous anatomical and physiological studies, correlation between morphology and functional connectivity, particularly in terms of inhibitory inputs, remains elusive. To compare excitatory and inhibitory synaptic inputs on individual SG neurones with morphology, we performed whole-cell recordings with Neurobiotin-filled-pipettes in horizontal slices from adult rat spinal cord with attached dorsal roots. Based on dendritic arborization patterns, four major cell types were confirmed: islet, central, radial and vertical cells. Dorsal root stimulation revealed that each class was associated with characteristic synaptic inputs. Islet and central cells had monosynaptic excitatory inputs exclusively from C-afferents. Islet cells received primary-afferent-evoked inhibitory inputs only from Aδ-fibres, while those of central cells were mediated by both Aδ- and C-fibres. In contrast, radial and vertical cells had monosynaptic excitatory inputs from both Aδ- and C-fibres and inhibitory inputs mediated by both fibre types. We further characterized the neurochemical nature of these inhibitory synaptic inputs. The majority of islet, central and vertical cells exhibited GABAergic inhibitory inputs, while almost all radial cells also possessed glycinergic inputs. The present study demonstrates that SG neurones have distinct patterns of excitatory and inhibitory inputs that are related to their morphology. The neurotransmitters responsible for inhibitory inputs to individual SG neurones are also characteristic for different morphological classes. These results make it possible to identify primary afferent circuits associated with particular types of SG neurone.     

Acute toxicity, inductive effects of liver enzymes and distribution in the liver of 1,2,3,7,8-pentachlorodibenzo-p-dioxin in rats

Acute toxicity, inductive effects of liver enzymes and liver persistency of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PenCDD) were compared with those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) using male Wistar rats. 1,2,3,7,8-PenCDD treatment at a dose of 0.1 mumol/kg resulted in significant depression of growth of rats from a day to 28 days after treatment. However, the effect was relatively less than that of 2,3,7,8-TCDD. On 5 days, similarly to 2,3,7,8-TCDD-treated group, liver hypertrophy and thymic atrophy were observed in 1,2,3,7,8-PenCDD-treated groups. In addition, 1,2,3,7,8-PenCDD showed potent 3-methylcholanthrene-type inducing ability. For example, the activities of benzo(a)pyrene 3-hydroxylase and DT-diaphorase were 25-fold and 10-fold of control, respectively. On 30 days, about 50% of the inductive effects on 5 days were maintained in both 1,2,3,7,8-PenCDD- and 2,3,7,8-TCDD-treated groups. Amount of 1,2,3,7,8-PenCDD distributed to the liver on 5 days was about 80-90% of dose and was about 1.5 times greater than that of 2,3,7,8-TCDD. About 50% of dose of 1,2,3,7,8-PenCDD remained even on 30 days after treatment. From these results, it is suggested that 1,2,3,7,8-PenCDD possessing the potent acute toxicity comparable to 2,3,7,8-TCDD and higher persistency in the liver might be more important than 2,3,7,8-TCDD in terms of the chronic toxicity.     

Changes in the nuclear uptake and retention of 3H-estrogen in gonadotrophs and lactotrophs as a function of age

A quantitative autoradiographic immunocytochemical study was performed in which the nuclear uptake and retention of 3H-estradiol (3H-E2) by luteinizing hormone (LH) and prolactin (PRL) cells was examined in 19-21-year-old baboons. 3H-E2 concentrating cells were found in all of the three lobes of the pituitary in varying percentages (38.7%, pars distalis; 17.1%, pars intermedia; 6.3%, pars nervosa). Approximately 80% of PRL cells and nearly 100% of LH cells were labeled. A count of the number of silver grains over nuclei revealed a marked variation of the accumulation of 3H-E2 by LH cells and to a lesser extent in PRL cells. These results suggest functional heterogeneity among LH and PRL cells. The present results are discussed in relation to the physiological state of old animals.     

Augmentation and inhibition of beta-adrenergic agonists-stimulated tissue cyclic AMP accumulation by alpha-adrenergic agonists in rat parotid gland

It was found that phenylephrine and methoxamine had two effects (one was inhibitory and the other was augmentative) on isoproterenol-stimulated cyclic AMP in rat parotid slices. The augmentation was abolished by alpha-adrenergic antagonists or by omission of calcium in the medium. Cyclic AMP accumulation by norepinephrine (NE) was significantly decreased by omission of calcium in the medium. Calmodulin antagonists, trifluoperazine and W-7, decreased NE-induced cyclic AMP accumulation, but another calmodulin antagonist, carmidazolium, did not. Phorbol ester such as 4 beta-phorbol 12-myristate, 13-acetate and phorbol 12, 13-dibutyrate, did not augment the effect of isoproterenol. These results suggest that although the influx of calcium is required in the alpha-adrenergic agonists-induced augmentation, calmodulin and protein kinase C may not be intermediates in this process. Calcium ions (10(-7) and 10(-6) M) slightly increased the activity of adenylate cyclase, but calcium (10(-6)-10(-4) M) dose-dependently inhibited the effect of isoproterenol. Therefore, calcium ions do not participate in the augmentation by directly modulating the activity of adenylate cyclase. The inhibitory effect was not affected by alpha-adrenergic antagonists. The activation of adenylate cyclase by isoproterenol was inhibited by phenylephrine with higher inhibition being obtained in lower concentrations of isoproterenol. Phenylephrine in the presence of isobutylmethylxanthine increased the amount of cyclic AMP and this effect was inhibited by propranolol, but not by phentolamine. [3H]-CGP 12177 binding of the parotid membrane was inhibited by alpha-adrenergic antagonists. These results suggest that the inhibitory effect of phenylephrine and methoxamine may be mediated by beta-adrenergic receptor.     

A rapid isolation technique of unmodified human T cells on a polystyrene resin column

An easy and rapid isolation technique of human T cells on a polystyrene resin particle column has been developed. The cells of the effluent fraction contained more than 90% sheep erythrocyte (SRBC) rosette-forming cells and less than 1% of cells bearing surface immunoglobulin (Ig) or peroxidase. The T cell (SRBC rosette-forming cells) recovery rate was 80%. The distribution of OKT antigen T cell subsets was essentially the same as that of T cells separated by rosette sedimentation. Cell functions such as tritiated thymidine uptake by T cells and helper activity in Ig production were also the same as that of T cells separated by SRBC rosette sedimentation. Natural killer-like activity of the T cells isolated by the present method increased more than that of T cells obtained by the conventional method. Moreover, it was free from functional modification which tends to result from stimulation such as by the SRBC antigen in the SRBC centrifugation method. The combination of a T cell population offered by the present method and B cells depleted of SRBC-binding B cells minimized background plaque formation and enabled us to quantify the plaque-forming cell number in an antigen-specific plaque-forming assay. Furthermore, these populations produced relatively pure interleukin 2 (IL 2) by stimulation of an autologous mixed lymphocyte reaction without any absorption of IL 2 produced in the same culture. It seemed to be useful to evaluate the ability of lymphocytes from normal individuals and patients to produce IL 2.     

Capsular Polysaccharide-Fimbrial Protein Conjugate Vaccine Protects against Porphyromonas gingivalis Infection in SCID Mice Reconstituted with Human Peripheral Blood Lymphocytes

The effect of immunization with either a Porphyromonas gingivalis fimbrial protein, a capsular polysaccharide, or a capsular polysaccharide-fimbrial protein conjugate vaccine were compared in hu-PBL-SCID mice. A significantly higher human immunoglobulin G antibody response and the highest degree of in vivo protection against bacterial challenge was observed in the group immunized with the conjugate vaccine. It was concluded that capsular polysaccharide-fimbrial protein conjugate from P. gingivalis could potentially be developed as a vaccine against periodontal infection by P. gingivalis.Porphyromonas gingivalis has been implicated as one of the major periodontal pathogens, and specific humoral and cell-mediated immune reactions to this organism have been demonstrated in periodontal diseases (13, 16). Attempts to induce protection against experimental infection with P. gingivalis by active immunization procedures have been studied by immunization with selected cell wall fractions, outer membrane proteins, and capsular polysaccharides (CPS) of P. gingivalis (8, 14). While most of these approaches afforded significant levels of protection (8, 14), problems such as maintaining functional levels of specific antibodies for extended periods of time (immune memory) (9), the multiple antigenicity of various pathogenic organisms, and the inability to activate T-cell-dependent immune responses (12) remain to be overcome. One strategy may be to develop a conjugation vaccine composed of CPS coupled with an outer membrane protein of P. gingivalis which can function as an immunodominant antigen as well as a carrier protein to activate T-cell-dependent immune responses.An additional area of improvement in vaccine strategies is the development of an adequate animal model system for simulating humanized antibody responses. Conventional animal models have disadvantages, since the animals may be qualitatively different from humans with respect to oral microbial environments and histological components in the development of periodontal lesions, and the nature of animal immune functions differs from that of human immune responses. Also, immunogenetic makeup (i.e., immunoglobulin [Ig] allotypes) and control over Ig class and subclass responses differ in animals and humans. Recently, mice with severe combined immunodeficiency (SCID) were identified (3, 15). The SCID mice lack functional T and B cells due to a mutation affecting the recombinase system that impairs the rearrangement of antigen receptor genes in both T and B cells. As postimmunization levels of IgG subclasses in vaccinees or in hu-PBL-SCID mice were closely associated with human Ig allotypes (5, 10, 11), we reconstituted the SCID mouse phenotype with human peripheral blood lymphocytes (PBL) whose Ig allotypes were positive for the phenotype fnb. As an extension of our previous experiments (5), we evaluated the protective effect of a newly developed polysaccharide-fimbrillin (FIM) protein conjugate vaccine with hu-PBL-SCID mice.Twenty-six SCID mice (C.B.-17-scid; Charles River Japan, Inc., Kanagawa, Japan) initially examined for IgG against P. gingivalis whole cells were reconstituted with 0.5 ml of human PBL (8 × 10⁷/ml) from periodontally healthy donors who were positive for the Ig phenotype fnb (either agfnb or axgfnb). Two weeks after reconstitution with human PBL, the expression of human Ig allotype markers was identified by the hemagglutination inhibition assay described previously (4).CPS of P. gingivalis 53977 was prepared by a modification of the method previously described (14). Briefly, bacterial cells were suspended in water (0.2 to 0.4 g [wet weight]/ml) and extracted with an equal volume of 90% phenol for 20 min at 65 to 68°C. The aqueous phase was obtained by centrifugation at 4,000 × g and dialyzed against distilled water with Spectrapor 1 tubing. The dialyzed solution was brought to 0.15 M sodium chloride, 4 mM MgCl₂, 1 mM CaCl₂, and pH 7.5 with Tris-HCl, treated with RNase A (0.04 mg/ml) and DNase I (0.01 mg/ml) (Sigma, St. Louis, Mo.) for 2 h at 37°C, treated with proteinase K (0.04 mg/ml) for 1 h at 60°C, dialyzed against dH₂O, and lyophilized. The lyophilized extract was dissolved in 0.05 M Tris-HCl buffer (pH 9.5) containing 0.3% deoxycholate and 0.001 M trisodium EDTA, applied to a column of Sephacryl S-400 HR (1.0 by 47 cm) (Pharmacia, Piscataway, N.J.), and eluted with the deoxycholate-containing buffer. Fractions were assessed for lipopolysaccharide and CPS by double immunodiffusion in agarose, for LPS contamination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and for protein contamination. Fractions containing only CPS were pooled, sodium chloride was added to 0.15 M, and CPS was precipitated with 4 volumes of 95% ethanol. The precipitates were isolated by centrifugation, dissolved, dialyzed, and lyophilized.Fimbriae of P. gingivalis 381 were purified as follows (17). Briefly, cells were harvested by centrifugation and suspended in 20 mM Tris-HCl (pH 7.4)–0.15 M NaCl–10 mM MgCl₂ by repeated pipetting. The suspension was agitated by magnetic stirrer for 30 min, and the supernatant was obtained after centrifugation at 8,000 × g for 20 min. Ammonium sulfate was added to 40% saturation, precipitated proteins were collected by centrifugation, and the precipitate was dissolved in 20 mM Tris-HCl (pH 8.0) and dialyzed against 20 mM Tris-HCl (pH 8.0). The dialysate was clarified by centrifugation at 8,000 × g for 20 min and applied to a column of DEAE-Sepharose CL-6B (1.5 by 16 cm) (Pharmacia) equilibrated with the above-described buffer. The column was washed with 20 mM Tris-HCl, pH 8.0, and eluted with a linear gradient of 0 to 0.3 M NaCl. The 43,000-molecular-weight protein (43K protein) band was not detected in fractions eluted after 0.17 M NaCl. Fractions containing the 43K protein were concentrated by ammonium sulfate precipitation and dialyzed against 3 mM Tris-HCl (pH 8.0) or 3 mM sodium bicarbonate (pH 8.0).The carboxylate group of the P. gingivalis CPS was conjugated to free amino residues of either cationic bovine serum albumin (CPS-BSA) or 43-kDa P. gingivalis fimbrillin (CPS-FIM) via a 1-ethyl-3-(dimethylaminopropyl)-carbodiimidide (EDC) intermediate. A total of 1.5 mg of CPS was dissolved in 0.5 ml of EDC conjugation buffer (Pierce, Rockford, Ill.), and 2 mg of BSA (SuperCarrier; Pierce) or 2 mg of fimbrial protein dissolved in 0.2 ml of water was mixed and added to the EDC (10 mg in 1 ml of water) and reacted for 2 h at room temperature. The mixture was separated from carbodiimidide by gel filtration on a Sephacryl S-300 column (1.0 by 10 cm) with 0.9 M sodium chloride and 0.083 M sodium phosphate, pH 7.2. Fractions were screened by immunodiffusion in agarose gel with rabbit antisera to CPS, fimbrial protein, or BSA.Only mice with no detectable amount of murine IgG against P. gingivalis were included in the study. Two weeks following PBL reconstitution, mice were examined for the expression of human Ig allotypes. Three of 26 mice were found to be leaky; a total of 23 mice were used in the experiment. Group I (n = 6) was immunized with FIM, group II (n = 6) was immunized with the CPS-BSA vaccine, group III (n = 6) was immunized with the CPS-FIM conjugate vaccine, and group IV (control, n = 5) was immunized with BSA. Each immunization procedure consisted of two intraperitoneal injections (at 2-week intervals) with 0.2 ml of immunogen adjuvant (Imject Alum; Pierce) mixture. The final amount of immunogen was 10 μg. Two weeks after the final immunization, mice were challenged by two dorsal subcutaneous injections (0.1 ml each) of whole P. gingivalis 53977 cells (1 × 10¹¹/ml) and evaluated for protective effects for 3 weeks based on the following criteria: general appearance, cachexia, weight loss, size and nature of localized abscess formation, development and size of secondary lesion, and death. Preimmune, postimmune (2 weeks following final immunization), and postinfection (3 weeks following infection) total IgG and IgG subclass antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) with an alkaline phosphatase assay system. Microtiter plates (96 well) were coated with 0.1 ml of antigen (10 μg/ml) diluted in 0.01 M phosphate buffer (pH 7.2). After overnight incubation at 4°C, the plates were washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween 20. A total of 0.05 ml of mouse serum samples diluted in PBS containing 0.1% Tween 20 was added to each well and incubated for 2 h at room temperature. The plate was washed three times with PBS containing 0.1% Tween 20, and then 0.1 ml of four mouse anti-human IgG subclasses (affinity-purified monoclonal antibody, γ-chain-specific, IgG1; 8c/6-39, IgG2; HP-6014, IgG3; HP-6050, IgG4; HP-6025; Sigma) diluted in PBS containing 0.1% Tween 20 were added to each well and incubated for 2 h at room temperature. After being washed three times with PBS containing 0.1% Tween 20, 0.05 ml of goat anti-mouse IgG (heavy- and light-chain specific, affinity purified, alkaline phosphatase conjugated; Calbiochem, Basel, Switzerland) diluted in PBS containing 0.1% Tween 20 were added to each well and incubated overnight at room temperature. After the plates were washed, 0.1 ml of nitrophenyl phosphate (1 mg/ml in diethanolamine buffer, pH 9.8) was added to each well and incubated for 60 min, and 0.1 ml of 3 N NaOH was added to stop the color reaction. For total IgG antibody measurements, goat anti-human IgG (affinity purified, γ-chain specific, alkaline phosphatase conjugated; Calbiochem) was used. Optical densities were plotted as a function of serum dilution factor, regression analysis was performed, and reciprocals of the serum dilution factors at the x axis intersection of an optical density of 0.2 were expressed in ELISA units for each sample. For the comparison of antibody levels between groups or intervals, Student’s t test was done.Except for those mice which were found to be leaky (n = 3), all mice (n = 23) expressed human Ig allotypes, either axgfnbt or agfnbt, according to the donors’ allotypes, confirming our previous observation (5). IgG and IgG subclass titers are summarized in Table ​Table1.1. Both postimmune and postinfection IgG levels against whole cells increased significantly compared to baseline values in all groups. IgG levels to whole cells in group III were significantly higher than those of groups I, II, or IV throughout the experimental period. In groups I and III, IgG4 subclass antibody titers were higher at the postinfection phase than at the postimmunization phase. Our previous studies have shown that early-onset periodontitis patients, whose haplotype fnb frequency was significantly higher than that of the race- and age-matched control group, had significantly higher IgG2 and IgG4 levels to P. gingivalis (4, 6). It was reasoned that the conversion of IgG1-restricted responses to IgG4-restricted responses with prolonged proteineous antigenic stimulation might be also responsible for the higher IgG4 subclass levels (1). IgG2 subclass responses were elevated to the polysaccharide antigen, while IgG1 responses were elevated to the fimbrial antigen, similar to previous results following bacterial infection or vaccination (2, 7, 10, 11). While all mice immunized with the conjugate vaccine survived the high-dose bacterial challenge (2 × 10¹⁰ cells of P. gingivalis 53977), one-third of the mice in the other two experimental groups and all of the mice in the control group died. The magnitude of the humoral antibody response was highest and the in vivo protective effect was greatest with minimal weight loss in group III (Tables ​(Tables11 and ​and2).2). This observation implies that through the use of a sophisticated conjugational vaccine incorporating two kinds of immunodominant antigens (i.e., outer membrane fimbrial protein and CPS), protection against P. gingivalis infection can be enhanced. Moreover, the SCID mice reconstituted with PBL from donors of the same IG allotypes provided a genetics-based simulation model for investigating humanized antibody responses to various vaccine formulas. By establishing T-cell hybridomas from hu-PBL-SCID mice, we are attempting to identify antigenic epitopes for T-cell clonal activation and to identify heavy- and light-chain variable gene usage of the antibodies from the conjugate vaccine.

TABLE 1

Baseline, postimmunization, and postinfection IgG and IgG subclass levels for each mouse group (ELISA unit ± standard deviation)