Induction of antitumor activity in macrophages by mycoplasmas in concert with interferon

Summary The in vitro growth of tumor cells infected with mycoplasmas was suppressed by macrophages pretreated with interferon (IFN), but the growth of mycoplasma-free tumor cells was not suppressed. Pretreatment of macrophages with IFN plus mycoplasmas or their soluble factors either simultaneously or sequentially, IFN first and mycoplasmas second, but not in the reverse order, was effective in activating macrophages to suppress the growth of mycoplasma-free tumor cells. Macrophages from C3H/HeJ mice (which respond only slightly to lipopolysaccharide) were activated by IFN plus mycoplasmas or their soluble factor, and their action was not influenced by the addition of a lipopolysaccharide-neutralizing agent, polymyxin B. These results suggest that the macrophage-activating agent in mycoplasmas does not mimic lipopolysaccharide. The administration of mycoplasmas plus IFN to mice with ascitic or solid tumors resulted in the reduction of tumor growth. The survival rate of tumor-bearing mice was improved by the administration of mycoplasmas, and this was synergistically enhanced by the addition of IFN. These results indicate (a) that mycoplasmas can be useful as a biological response modifier, and (b) that care should be taken to prevent contamination with mycoplasmas in experiments on macrophage activation.     

Kidney and liver metallothioneins in rats after administration of an organic compound

Intubation of rats with alpha-mercapto-beta-(2-furyl)-acrylic acid (MFA) for 5 days at 50 mg/kg caused a 7-fold increase in kidney copper concentration, a 2-fold increase in kidney zinc concentration, and a 20% increase in liver zinc concentration. The proteins which bound the increased metals were purified and identified as metallothioneins by their amino acid compositions. Two isoforms were isolated from each organ. Renal thioneins appeared identical to counterpart hepatic apoproteins, but the former bound Cu and Zn in a 2:1 mole ratio and the latter bound only Zn. Kidney contained over 10 times more metallothionein per g of tissue than did liver. In rats previously administered MFA, injection of cadmium sulfate resulted in rapid displacement of liver metallothionein-bound Zn by Cd under conditions where minimal metallothionein was found in Cd-dosed animals not administered MFA. We conclude that MFA induces metallothionein biosynthesis in kidney and liver of normal rats; this is a novel effect for an organic compound.     

Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice

Blank, Sally E., T. Bucky Jones, Eric G. Lee, C. JayneBrahler, Randle M. Gallucci, Marne L. Fox, and Gary G. Meadows. Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice. J. Appl.Physiol. 83(3): 845-850, 1997.This study wasdesigned to investigate the effects of moderate-intensity endurancetraining on basal natural killer (NK) cell cytolytic activity in murinesplenocytes that were enriched for1)NK1.1⁺ cells or2) macrophages andNK1.1⁺ cells. Mice were assignedto sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn)groups. Splenocyte number, the percentages ofNK1.1⁺, large granular lymphocytes(NK1.1⁺, LGL-1⁺),and other subpopulations did not change in Trn mice. Approximately 70%of cells enriched for NK1.1⁺expressed this surface antigen. Lytic units (LU) expressed per LGL-1⁺ cell were significantlylower in Trn [83.9 ± 3.2 (SE)] compared with Sed (109.5 ± 7.5) and TM (101.3 ± 6.4) groups. When macrophages remainedin the in vitro assay, LU perLGL-1⁺ cell did not differ acrossgroups. The results indicate that highly enrichedNK1.1⁺ cells from Trn mice hadlower NK cell activity compared with Sed mice. No differences in NKcell activity were observed when cells were enriched forNK1.1⁺ cells and macrophages.These findings support the hypothesis that macrophage modulation of NKcells may be one mechanism contributing to augmented basal NK cellactivity in endurance-trained individuals.

     

Systemic activation of tumoricidal properties in mouse macrophages and inhibition of melanoma metastases by the oral administration of MTP-PE, a lipophilic muramyl dipeptide

The purpose of these studies was to determine whether the oral administration of a lipophilic analog of muramyl dipeptide, MTP-PE, can produce in situ activation of tumoricidal properties in mouse macrophages. MTP-PE was dissolved in a phosphate-buffered saline to produce micelles. Single or multiple oral administrations of MTP-PE produced tumoricidal activation in both lung and peritoneal macrophages. This was in direct contrast to the i.v. or i.p. administrations of MTP-PE incorporated in liposomes, which produced activation in only lung or only peritoneal macrophages, respectively. The distribution and fate of [3H]-labeled MTP-PE subsequent to oral administration revealed that MTP-PE was found in various organs independent of reticuloendothelial activity. Finally, the repeated twice-weekly oral administrations of MTP-PE inhibited lung and lymph node metastasis in C57BL/6 mice by syngeneic B16 melanoma cells. The oral administration of MTP-PE, however, was not effective in eradicating well-established melanoma metastases. We conclude that the oral administration of a lipophilic muramyl dipeptide produces systemic activation of macrophages. The feasibility of enhancing host defense against infections and cancer by the oral administration of an immunomodulator has obvious clinical advantages.     

Alpha interferon and HIV infection cause activation of human T cells in NSG-BLT mice

The development of small animal models for the study of HIV transmission is important for evaluation of HIV prophylaxis and disease pathogenesis. In humanized bone marrow-liver-thymus (BLT) mice, hematopoiesis is reconstituted by implantation of human fetal liver and thymus tissue (Thy/Liv) plus intravenous injection of autologous liver-derived hematopoietic stem progenitor cells (HSPC). This results in reconstitution of human leukocytes in the mouse peripheral blood, lymphoid organs, and mucosal sites. NOD-scid interleukin-2 receptor-negative (IL-2Rγ(-/-)) (NSG)-BLT mice were inoculated intravaginally with HIV and were monitored for plasma viremia by a branched DNA assay 4 weeks later. T-cell activation was determined by expression of CD38 and HLA-DR on human CD4(+) and CD8(+) T cells in mouse peripheral blood at the time of inoculation and 4 weeks later. Additional BLT mice were treated with human alpha interferon 2b (IFN-α2b) (intron A) and assessed for T-cell activation. Productive HIV infection in BLT mice was associated with T-cell activation (increases in CD38 mean fluorescence intensity and both the frequency and absolute number of CD38(+) HLA-DR(+) T cells) that correlated strongly with plasma viral load and was most pronounced in the CD8(+) T-cell compartment. This T-cell activation phenotype was recapitulated in NSG-BLT mice treated with intron A. HIV susceptibility correlated with the number of HSPC injected, yet a number of mice receiving the Thy/Liv implant alone, with no HSPC injection, were also susceptible to intravaginal HIV. These results are consistent with studies linking T-cell activation to progressive disease in humans and lend support for the use of NSG-BLT mice in studies of HIV pathogenesis.     

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.     

Induction of activated macrophages by intraperitoneal injection of mitomylin C in mice

Summary The host cellular response to IP injection of mitomycin C was studied in C3H/HeN mice. As assessed by in vitro cytolysis assay using ¹²⁵I-iododeoxyuridine-labelled tumour target cells, mitomycin C-induced peritoneal macrophages showed the maximum tumouricidal activity 4 days after the IP injection. The tumouricidal activity was dependent on the dose of mitomycin C injected and it was detectable against syngeneic, allogeneic and xenogeneic tumour target cells. In addition, these tumouricidal macrophages were found to be augmented in functions of both incorporation of 2-deoxy-D-glucose and phagocytosis of sheep red blood cells. Among the other anti-cancer drugs, which were used at a dose of three-fifths of LD₅₀, only adriamycin (7.5 mg/kg) was capable of inducing activated macrophages as much as mitomycin C (3 mg/kg). Cyclophosphamide (225 mg/kg), methotrexate (60 mg/kg) and vincristin (1.5 mg/kg) were able to augment incorporation of 2-deoxy-D-glucose and phagocytosis of sheep red blood cells, but not tumouricidal actvity. Differential cytolysis assay was performed for two cell lines of P 388 tumour target cells, the mitomycin C-sensitive original cell line and the mitomycin C-resistant subline, demonstrating no significant difference in macro-phage-mediated tumour cell lysis between these cell lines. Based on these results, it was concluded that mitomycin C, when injected IP induced activated macrophages in the peritoneal cavity. A better understanding of the effect of anti-cancer drugs on macrophage tumouricidal activity may be useful in designing more effective local chemotherapy for malignant peritoneal effusions.     

Development of intestinal intraepithelial lymphocytes, NK cells, and NK 1.1+ T cells in CD45-deficient mice

The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6(-/-) mouse. As NK1.1(+) CD3(+) (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9(-/-)) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45-deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45-deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9(-/-) mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8(+)) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-gamma. In the absence of CD45, a significant population of extrathymically derived CD8alphaalpha(+) iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8(+) NK-T cells, NK cells, or CD8alphaalpha(+) iIELs, but is essential for TCR-mediated function and cytokine production.     

Prevention of pathogenic Escherichia coli infection in mice and stimulation of macrophage activation in rats by an oral administration of probiotic Lactobacillus casei I-5

Lactobacillus casei I-5 isolated from an alcohol fermentation broth enhanced immunity and prevented pathogenic infection as a probiotic. Mice fed with I-5 cells for 11 days prior to an intraperitoneal challenge with pathogenic Escherichia coli Juhl exhibited a high survival rate compared with the control group. Rats fed with I-5 cells for 10 days significantly increased the phagocytosis of peritoneal macrophages. In a cell culture system employing peritoneal macrophages from rats, the I-5 administration activated NF-kappaB stimulated by LPS. It also enhanced LPS-stimulated IL-12 and TNF-alpha production, but not IL-6 production. These results show that L. casei I-5 effectively prevented infection by pathogenic E. coli possibly through the activation of peritoneal macrophages. The strain would be useful to prevent pathogenic microbial infections in humans and farm animals.     

Induction of interferon and erythropoietic differentiation in cells transformed by Friend virus.

Two lines of Friend virus (FV)-transformed mouse spleen cells have been analyzed in respect to their interferon production capacity: neither F4 cells, which liberate infectious FV when kept under tissue culture conditions, nor the thymidine kinase-deficient B8 cells, which do not produce significant amounts of FV, release detectable amounts of autogenous interferon into cell supernatants. However, interferon is produced in these cells in amounts comparable to that in L-929 cells when interferon induction is initiated with UV-inactivated Newcastle disease virus. Conversely poly(I)-poly(C), a potent interferon inducer in L-929 cells, proved ineffective in this capacity in F4 or B8 cells. When erythropoietic differentiation is induced in these cells by dimethyl sulfoxide, no autogenous interferon production occurs, but with NDV-induction a four- to fivefode increase of interferon production is observed. A similar elevation of interferon production is achieved during 5-bromodeoxyuridine stimulation of differentiation in the thymidine kinase-deficient B8 cells. The refractiveness against poly(I)-poly(C) displayed in unstimulated cells is not overcome at any stage of differentiation, indicating major differences of Newcastle disease virus and poly(I)-poly(C) induction mechanisms.     

Enhancement of eicosanoid synthesis in mouse peritoneal macrophages by the organic mercury compound thimerosal

Availability of the common precursor arachidonic acid represents the fundamental prerequisite of the cellular eicosanoid synthesis. The amount of free arachidonic acid is regulated not only by phospholipases, which liberate this polyunsaturated fatty acid from lipid pools, but also by the reacylating enzyme acylCoA:lysophosphatide acyltransferase. We have previously shown (Goppelt-Strübe, G., C.-F. K?rner, G. Hausmann, D. Gemsa, and K. Resch. Control of Prostanoid Synthesis: Role of Reincorporation of Released Precursor Fatty Acids. Prostaglandins 32:373. 1986.) that the organic mercury compound thimerosal in murine peritoneal macrophages inhibits arachidonic acid reincorporation into cellular lipids, thereby leading to an enhanced prostanoid synthesis. In this report we show that the production of leukotriene C4 was also increased after the addition of thimerosal to mouse peritoneal macrophages in a time and dose dependent manner. Concomitantly, thimerosal led to a significant rise of the intracellular calcium concentration as measured by fura-2 fluorescence. Simultaneous addition of thimerosal and indomethacin or exogeneous arachidonic acid to the cells resulted in a synergistic enhancement of leukotriene C4 synthesis. On the other hand, another sulfhydryl group blocking agent, ethacrynic acid, was found to be ineffective in increasing leukotriene C4 levels even in combination with exogeneous arachidonic acid. Thimerosal therefore provides a helpful tool in studying the basic regulatory mechanisms of the cellular leukotriene synthesis.     

Enhancement of eicosanoid synthesis in mouse peritoneal macrophages by the organic mercury compound thimerosal

Availability of the common precursor arachidonic acid represents the fundamental prerequisite of the cellular eicosanoid synthesis. The amount of free arachidonic acid is regulated not only by phospholipases, which liberate this polyunsaturated fatty acid from lipid pools, but also by the reacylating enzyme acylCoA:lysophosphatide acyltransferase. We have previously shown (Goppelt-Strübe, G., C.-F. Körner, G. Hausmann, D. Gemsa, and K. Resch. Control of Prostanoid Synthesis: Role of Reincorporation of Released Precursor Fatty Acids. Prostaglandins : 373. 1986.) that the organic mercury compound thimerosal in murine peritoneal macrophages inhibits arachidonic acid reincorporation into cellular lipids, thereby leading to an enhanced prostanoid synthesis. In this report we show that the production of leukotriene C₄ was also increased after the addition of thimerosal to mouse peritoneal macrophages in a time and dose dependent manner. Concomitantly, thimerosal led to a significant rise of the intracellular calcium concentration as measured by fura-2 fluorescence. Simultaneous addition of thimerosal and indomethacin or exogeneous arachidonic acid to the cells resulted in a synergistic enhancement of leukotriene C₄ synthesis. On the other hand, another sulfhydryl group blocking agent, ethacrynic acid, was found to be ineffective in increasing leukotriene C₄ levels even in combination with exogeneous arachidonic acid. Thimerosal therefore provides a helpful tool in studying the basic regulatory mechanisms of the cellular leukotriene synthesis.     

Induction of type 1 interferon receptor by zinc in U937 cells

This study aims to determine whether zinc enhances interferon (IFN)-α activity in U937 cells. Type 1 IFN2 receptor (IFNAR2) protein in U937 cells was measured by flow cytometry. After 24 h of exposure to zinc chloride or polaprezinc (a chelate of zinc and l-carnosine) at concentrations ranging from 50 to 200 μM, histograms showing anti-IFNAR2 antibody-positive cells shifted to a higher FITC intensity. Zinc chloride and polaprezinc increased IFNAR2 mRNA levels approximately 30% and 40%, respectively, compared to the control. l-Carnosine alone did not alter IFNAR2 mRNA or protein levels. Cellular levels of 2′–5′ oligoadenylate synthetases (OAS) were markedly increased by IFN-α, and the increase was significantly accelerated by polaprezinc. However, polaprezinc alone did not increase 2′–5′OAS levels. The finding suggests that zinc, especially polaprezinc, enhances the expression of INFAR2 in U937 cells, thereby inducing production of the anti-viral protein 2′–5′OAS.     

Induction of self-tolerance and enhanced stress protein synthesis in L-132 cells by cadmium chloride and by hyperthermia

The effect of heat shock or cadmium treatment on protein synthesis and cell survival in L-132 cells has been examined. After cadmium treatment, the synthesis of a polypeptide of Mr 68000 (P68) was greatly enhanced over that of untreated cells. Besides P68 the synthesis of another polypeptide of Mr 89000 (P89) was also enhanced in heat-shocked cells. Both heat shock and cadmium treatment induced self-tolerance. The kinetics of the synthesis of induced polypeptides correlated well with the development of self-tolerance. The patterns of peptide maps obtained after partial proteolytic digestion from P68 induced in cadmium-treated and heat-shocked cells were virtually identical. However, neither heat-shocked cells did not confer cadmium tolerance nor did cadmium-treated cells induce thermotolerance.     

Production of gamma interferon by human T and null cells and its regulation by macrophages

Human mononuclear subpopulations were tested for the capacity to produce interferon after mitogenic stimulation with protein A from Staphylococcus aureus. Mononuclear cells were separated into highly enriched macrophage, T-, B-, and null-cell subpopulations by Sephadex G-10 adherence, anti-human IgG F(ab′) two-column chromatography, and rosetting with sheep erythrocytes. Interferon (IFN) production was observed in both T- and null-cell preparations, but not in macrophage or B-cell preparations. Physicochemical and antigenic characterization of IFN from T- and null-cell preparations showed that both mononuclear subpopulations produced gamma IFN (IFNγ). Regulatory studies showed that IFNγ production was differentially regulated by macrophages. Macrophage addition to T lymphocytes augmented both cellular proliferation and IFNγ production, whereas macrophage addition to null cells suppressed IFNγ production and had no effect on the minimal proliferative response observed for these cells.     

Induction of oral tolerance in mice by continuous feeding with beta-lactoglobulin and milk

We examined the immune response of mice to beta-lactoglobulin (BLG), a potent milk allergen, after continuous feeding of a BLG solution or milk instead of drinking-water. Strong suppression of the anti-BLG antibody response and antigen-specific T cell response were observed in mice fed BLG and milk. Although the profile of the antibody specificity to BLG peptides in mice fed BLG or milk was different from the control, the dominant determinants were still recognized and a limited number of new recognition sites appeared by BLG or milk feeding. These findings suggest that continuous feeding with BLG or milk not only induced tolerance in the periphery, but also priming. In terms of the antigen-specific IgG subclass response, the production of both IgG1 and IgG2a was significantly reduced. The cytokine secretion of interleukin (IL)-2, IFN-gamma and IL-10 was also reduced in the culture supernatants of lymph node cells from the mice fed BLG. The results indicate that the continuous feeding of BLG or milk induces suppression of both Th1- and Th2-dependent responses. This may reflect a state of oral tolerance induced by food ingestion.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Elimination of porcine hemopoietic cells by macrophages in mice.

The difficulty in achieving donor hemopoietic engraftment across highly disparate xenogeneic species barriers poses a major obstacle to exploring xenograft tolerance induction by mixed chimerism. In this study, we observed that macrophages mediate strong rejection of porcine hemopoietic cells in mice. Depletion of macrophages with medronate-encapsulated liposomes (M-liposomes) markedly improved porcine chimerism, and early chimerism in particular, in sublethally irradiated immunodeficient and lethally irradiated immunocompetent mice. Although porcine chimerism in the peripheral blood and spleen of M-liposome-treated mice rapidly declined after macrophages had recovered and became indistinguishable from controls by wk 5 post-transplant, the levels of chimerism in the marrow of these mice remained higher than those in control recipients at 8 wks after transplant. These results suggest that macrophages that developed in the presence of porcine chimerism were not adapted to the porcine donor and that marrow-resident macrophages did not phagocytose porcine cells. Moreover, M-liposome treatment had no effect on the survival of porcine PBMC injected into the recipient peritoneal cavity, but was essential for the migration and relocation of these cells into other tissues/organs, such as spleen, bone marrow, and peripheral blood. Together, our results suggest that murine reticuloendothelial macrophages, but not those in the bone marrow and peritoneal cavity, play a significant role in the clearance of porcine hemopoietic cells in vivo. Because injection of M-liposomes i.v. mainly depletes splenic macrophages and liver Kupffer cells, the spleen and/or liver are likely the primary sites of porcine cell clearance in vivo.     

NK1, NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues,and in bone and dental hard tissue cells

The distribution of the tachykinin receptors neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3), and the calcitonin gene-related peptide-1 (CGRP1) receptor were examined in rat teeth and tooth-supporting tissues by immunohistochemical methods and light and confocal microscopy. Western blot analysis was performed to identify the NK1- and the CGRP1-receptor proteins in the dental pulp. The results showed that odontoblasts and ameloblasts, cementoblasts and cementocytes, osteoblasts and osteocytes are all supported with the tachykinin receptors NK1 and NK2, but a distinct, graded cellular labeling pattern was demonstrated. The ameloblasts were also positive for CGRP1 receptor. Blood vessels in oral tissues expressed the tachykinin receptors NK1, NK2 and NK3, and the CGRP1 receptor. Both gingival and Malassez epithelium were abundantly supplied by NK2 receptor. Pulpal and periodontal fibroblasts demonstrated NK1 and NK2 receptors. Western blot analysis identified both the NK1- and the CGRP1-receptor proteins in the dental pulp. These results clearly indicate that the neuropeptides substance P, neurokinin A, neurokinin B and CGRP, released from sensory axons upon stimulation, directly modulate the function of the different types of bone and dental hard tissue cells, and regulate functions of blood vessels, fibroblasts and epithelial cells in oral tissues.