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Article

Histamine Deficiency Inhibits Lymphocyte Infiltration in the Lacrimal Gland of Aged Mice

by
Hirotada Otsuka
1,2,*,
Yusuke Tsunoyama
3,
Miki Koh
1,
Satoshi Soeta
1 and
Naoko Nonaka
2
1
Laboratory of Veterinary Anatomy, Nippon Veterinary and Animal Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan
2
Department of Oral Anatomy and Developmental Biology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
3
Zephyr Animal Hospital, 21-3 Namikicho Hachioji-Shi, Tokyo 193-0831, Japan
*
Author to whom correspondence should be addressed.
Lymphatics 2025, 3(4), 48; https://doi.org/10.3390/lymphatics3040048
Submission received: 7 August 2025 / Revised: 26 September 2025 / Accepted: 16 December 2025 / Published: 17 December 2025
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Abstract

Aging is associated with chronic low-grade inflammation of exocrine glands, such as the lacrimal glands. Histamine, synthesized by histidine decarboxylase (HDC), is implicated in immune modulation; however, its role in age-related lacrimal gland inflammation remains unclear. To explore the role of histamine in age-related lacrimal gland inflammation, we compared wild-type and histidine decarboxylase knockout (HDC-KO) C57BL/6 mice at 6 weeks and 12 months of age (10 males and 10 females in each group). Histological and immunohistochemical analyses were performed to assess lymphocytic infiltration, mast cells, and the expression of cytokines and adhesion molecules. Gene expression levels were quantified using reverse transcriptase quantitative PCR (RT-qPCR). Aged wild-type mice showed significant upregulation of mRNA transcription of HDC and histamine H1 receptor, along with increased infiltration of B220-positive B cells and CD3-positive T cells in the lacrimal gland. The mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and ICAM-1 were elevated with age, whereas these changes were attenuated in HDC-KO mice. The mRNA expression of PPARγ, an anti-inflammatory factor, was upregulated in the aged HDC-KO mice. Mast cell numbers increased with age but did not differ according to sex. These findings suggest that histamine, via HDC and H1 receptor signaling, contributes to age-associated lacrimal gland inflammation by enhancing cytokine and ICAM-1 expression. HDC deficiency suppresses this inflammatory response, potentially through the upregulation of PPARγ. Thus, histamine may be a key mediator of age-related inflammation in the lacrimal gland and a potential therapeutic target.

1. Introduction

Histamine is a biologically active amine synthesized by histidine decarboxylase (HDC) that plays diverse roles in various physiological processes, including inflammatory responses, immune regulation, allergic reactions, and neurotransmission [1,2,3]. Histamine exerts its local effects in a paracrine or autocrine manner through four distinct receptors (H1R, H2R, H3R, and H4R), thereby inducing classical immediate-type hypersensitivity responses [4,5,6,7]. Additionally, histamine has been shown to directly modulate immune cell activity via receptor stimuli [1].
HDC is highly expressed in immune-related organs, such as the spleen and bone marrow, and its expression is induced by pro-inflammatory cytokines (e.g., interleukin-1) and lipopolysaccharide (LPS) stimulation [8,9]. In our previous studies using HDC-deficient mice, we demonstrated that the absence of histamine significantly attenuated the induction of inflammatory cytokines in response to nitrogen-containing bisphosphonate (NBP), a compound known to inhibit bone resorption [10]. Histamine contributes to the promotion of inflammatory responses through bidirectional crosstalk with immune cells. Specifically, histamine activates histamine receptors expressed on immune cells, leading to the induction of pro-inflammatory cytokines such as TNFα and IL-1β [4,11,12]. These cytokines, in turn, enhance histamine synthesis, thereby establishing a positive feedback loop that sustains inflammation [13,14]. However, the relationship between HDC and chronic inflammation is unclear.
Age-related upregulation of HDC and/or histamine expression has been documented in various tissues [8,15,16,17]. Moreover, numerous studies have demonstrated a correlation between elevated histamine levels and inflammation [18]. With advancing age, tissues undergo persistent low-grade inflammation, a process commonly referred to as “inflammaging” [19,20]. We found that age-related lymphocytic infiltration into the submandibular glands was markedly reduced in HDC-deficient mice [21]. Considering that HDC expression increases in response to inflammation and aging, elevated histamine levels may contribute to age-associated tissue alterations and chronic inflammation. However, it remains unclear whether this histamine elevation directly drives inflammatory responses in aging tissues or represents a secondary phenomenon accompanying structural or functional degeneration.
The salivary and lacrimal glands undergo functional decline with aging and autoimmune diseases, such as Sjögren’s syndrome [22,23]. Secretary dysfunction of the salivary and lacrimal glands is caused by autoantibody deposition, which leads to acinar cell destruction in autoimmune conditions, causing dry eye and xerostomia [24,25]. Similar pathological manifestations have been documented in aged mice, characterized by antibody deposition and inflammation across multiple tissues, including the salivary and lacrimal glands [26]. In our previous study, we identified prominent age-associated alterations in the submandibular gland that were markedly attenuated in histidine decarboxylase (HDC)-deficient mice [21]. Symptoms such as age-related dry eye and mouth are commonly attributed to reductions in glandular secretion capacity; however, the molecular mechanisms underlying these changes, particularly those involving inflammation, remain poorly understood.
In this study, we aimed to elucidate the relationship between age-associated changes in HDC expression and inflammatory or functional alterations in the lacrimal glands using HDC-deficient mice as a model to investigate the potential role of histamine in age-related tissue pathology.

2. Results

2.1. Age-Related Alterations in the Expression of Histidine Decarboxylase and Histamine Receptors in the Lacrimal Gland

Quantitative real-time reverse transcription PCR (RT-qPCR) data demonstrated significant upregulation of HDC mRNA expression in aged (12 months of age) compared to young adult (6 weeks of age) wild-type mice (Figure 1A). HDC mRNA expression significantly increased with age in both male and female mice (Figure 1B). Additionally, no sex-related differences in HDC mRNA expression were observed at 12 months of age (Figure 1B). The results revealed a significant increase in H1 receptor mRNA expression with age, whereas no significant differences were observed in the mRNA expression levels of the H2 and H4 receptors between young and aged mice (Figure 1C–E).

2.2. Isolation of Mast Cells from Lacrimal Glands of 6-Week and 12-Month-Old Wild-Type Mice

In toluidine blue (TB)-stained sections, a small number of mast cells displaying a metachromatic reaction were observed in the stroma of the lacrimal glands of 6-week-old wild-type mice (Figure 2A). In 12-month-old wild-type mice, mast cells were also observed in the stroma of the lacrimal glands (Figure 2B). Furthermore, the number of mast cells per unit area was significantly increased at 12 months of age compared to that at 6 weeks of age (Figure 2C). However, no sex-related differences were observed in mast cell density at 12 months of age (Figure 2D).

2.3. Age-Related Morphological Changes in Lacrimal Gland Tissue

In HE-stained sections, lymphocytic infiltration within the lacrimal gland tissue was observed in aged wild-type mice (Figure 3A,B). However, no evidence of acinar cell death or fatty degeneration was observed. To assess the impact of HDC deficiency, lacrimal gland tissues from young and aged HDC-KO mice were examined (Figure 3C,D). In contrast to that, in wild-type mice, lymphocytic infiltration was minimal in the lacrimal glands of aged HDC-KO mice (Figure 3B,D). Furthermore, comparison of the number of infiltrating cells per unit area revealed a significant decrease in HDC-KO mice compared to wild-type controls at 12 months of age (Figure 3E). When stratified by sex, no statistically significant differences were observed between males and females in the aged wild-type and HDC-KO groups (Figure 3F).

2.4. Identification of Infiltrating Cells in the Lacrimal Glands of Aged Wild-Type Mice

Significant cellular infiltration observed in the lacrimal glands of 12-month-old wild-type mice consisted of abundant CD3-positive T-lymphocytes and comprised both CD4-positive and CD8α-positive subsets (Figure 4A–C). The majority of infiltrating cells were B220-positive B-lymphocytes (Figure 4D). In addition, CD11c-positive dendritic cells and F4/80-positive macrophages were detected (Figure 4E,F). Notably, CD11c-positive cells were localized within lymphocytic aggregates, whereas F4/80-positive cells were predominantly found at the periphery of these aggregates (Figure 4E,F).

2.5. The Age-Related Changes of Inflammatory Associated Cytokines Expression in the Lacrimal Gland

In wild-type mice, the mRNA expression levels of TNFα and IL-1β significantly increased with age (Figure 5A,B). Furthermore, a significant age-related increase in TNFα mRNA expression was observed in HDC-KO mice (Figure 5A). When comparing aged wild-type and HDC-KO mice, the mRNA expression levels of both TNFα and IL-1β were significantly higher in wild-type mice than in HDC-KO mice (Figure 5A,B). In contrast, the mRNA expression of IL-6 was undetectable in either genotype during young adulthood (Figure 5C). In aged mice, IL-6 mRNA expression was lower in HDC-KO mice than in wild-type control mice (Figure 5C). The mRNA expression of peroxisome proliferator-activated receptor gamma (PPARγ) did not exhibit significant age-related changes in wild-type mice. However, its expression increased with age in HDC-KO mice (Figure 5D).

2.6. Changes in Intercellular Adhesion Molecule 1 (ICAM-1) Expression

Immunohistochemical analysis revealed the presence of ICAM-1–positive acini, showing immunoreactivity on the basal side of the acinar cells in both aged wild-type and HDC-KO mice (Figure 6A,B). Furthermore, a quantitative assessment of ICAM-1 mRNA levels revealed a significant age-associated increase in wild-type mice but not in HDC-KO mice (Figure 6C). Notably, when comparing genotypes, the mRNA expression level of ICAM-1 was lower in aged HDC-KO mice than in aged wild-type mice (Figure 6C).

3. Discussion

We demonstrated that the mRNA expression of HDC and H1 in the lacrimal gland increased with age, whereas no significant changes were observed in the mRNA expression of H2 or H4 receptors (Figure 1A,C–E). The observed upregulation of HDC in our study is likely attributable to an increase in mast cell populations, consistent with our previous findings in the submandibular gland (Figure 2C) [21]. H1 receptor stimulation has been reported to enhance T cell migration, shift the Th1/Th2 balance toward a Th1-dominant state, and is associated with the release of cytokines and adhesion molecules, such as TNF-α, IL-6, IL-8, and ICAM-1 [4,11,27,28,29,30]. These findings suggest that histamine signaling via HDC and H1 contributes to age-related lacrimal gland inflammation, possibly through a Th1-biased immune response.
Our findings further demonstrated that HDC deficiency suppressed age-related inflammatory cell infiltration in the lacrimal glands, consistent with our previous observations in salivary glands (Figure 3) [21]. In contrast to the lymphoid tissues normally present in the conjunctiva and nasolacrimal duct, infiltration into the functional parenchyma of the lacrimal gland, as observed here, has not been previously reported [31]. Given that lymphocytic infiltration in Sjögren’s syndrome model mice is associated with reduced tear secretion, the present results suggest that similar immune mechanisms may contribute to the functional impairment of the lacrimal gland [32]. This finding indicates a pathological inflammatory process distinct from infiltration by preexisting lymphoid tissue. Additionally, we did not observe any significant sex-related differences in HDC mRNA expression and the extent of inflammatory cell infiltration, although sex hormones are known to influence both the prevalence of Sjögren’s syndrome and tear secretion function [33]. Although it appears to resemble a model of Sjögren’s syndrome, sex-related differences have not been fully elucidated.
We observed that the mRNA expression levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly elevated in aged wild-type mice (Figure 5A–C). In contrast, HDC-KO mice exhibited a less pronounced increase in these cytokines than wild-type mice (Figure 5A–C). Previous studies have indicated mutual reinforcement between histamine and cytokines, and our data extend this concept to age-related changes in the lacrimal gland [11,13]. Furthermore, the anti-inflammatory factor PPARγ was upregulated compared to pro-inflammatory cytokines in HDC-KO mice (Figure 5D). PPARγ inhibits mast cells and inflammatory responses by inhibiting cytokines and exerting protective effects against aging [34,35]. Therefore, it is possible that they are involved in the suppression of histamine-induced inflammation.
In addition, ICAM-1 expression was significantly higher in aged wild-type mice than in HDC-KO mice (Figure 5A,B). Previous studies have suggested an association between ICAM-1 expression and the pathogenesis of Sjögren’s syndrome [36]. ICAM-1 plays a crucial role in immune activation, which is associated with autoimmune pathogenesis and is induced by pro-inflammatory cytokines, including IL-1 and IL-6 [37,38,39]. Our results suggest that histamine signaling via H1 may promote ICAM-1 upregulation, thereby exacerbating inflammation.
Importantly, our findings indicate that the modulation of histamine metabolism may represent a promising therapeutic strategy for Sjögren’s syndrome. Pharmacological inhibition of HDC activity or histamine receptor signaling could potentially attenuate inflammatory cell infiltration, thereby preserving lacrimal gland function. Furthermore, the integration of histamine-targeted interventions with existing treatment modalities may facilitate the development of more effective and personalized therapeutic approaches for patients with Sjögren’s syndrome and related forms of dry eye disease.
However, this study has some limitations. We did not examine the cellular localization of HDC or histamine receptors, nor did we evaluate T cell subsets or hormone levels, which may influence sex-related effects in this study. Furthermore, our analyses were limited to mRNA expression; therefore, validation at the protein level and functional assessment of tear secretion are required in future studies to confirm these findings.

4. Materials and Methods

4.1. Animals

HDC-KO mice were provided by the School of Engineering, Tohoku University [40]. In all experiments, HDC-KO and wild-type mice littermates (C57BL/6J background) were obtained by mating heterogeneous mice, unless otherwise specified. Mice were genotyped using PCR with tail DNA [41]. For all experiments, male and female mice were housed in groups of five per cage and maintained under specific pathogen-free conditions (temperature 25 °C, 12 h light/dark cycle), with free access to food and water, and were used at 6 weeks (young) and 12 months (aged) of age, with separation according to sex, genotype, and age. The mice were fed a histamine-free diet (Oriental Kobo, Tokyo, Japan) to avoid the effects of histamine throughout the experimental period. Twenty mice (10 males and 10 females) from each genotype and age group were used in this study. Samples for histological analysis and RT-qPCR were obtained from the same mice.
All experiments were performed with the approval of the Animal Care Committee of Nippon Veterinary and Life Science University (Permit Numbers: 2024 K-1 and 2025 K-1), following all applicable international, national, and institutional guidelines for the care and use of animals.

4.2. Antibodies

The following monoclonal antibodies were used in this study: purified anti-mouse B220 (B-lymphocyte marker) rat antibody (BD Pharmingen, San Diego, CA, USA); purified anti-mouse F4/80 (macrophage marker) rat antibody (Bio-Rad, Hercules, CA, USA); anti-mouse CD3e (T-lymphocyte marker), CD4 (helper T cell marker), CD8α (cytotoxic T cell marker), and CD11c (dendritic cell marker) rabbit monoclonal antibodies (Cell Signaling Technology, Danvers, MA, USA); anti-mouse ICAM-1 (adhesion molecule) rabbit polyclonal antibody (Proteintech, Rosemont, IL, US); HRP-conjugated goat anti-rat IgG antibody (Nichirei, Tokyo, Japan); HRP-conjugated goat anti-rabbit IgG antibody; and normal goat serum (Dako, Agilent, CA, USA).

4.3. Tissue Preparation for Histology and Immunohistochemical Staining

Tissue samples were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). Tissue samples were routinely processed and embedded in paraffin. Paraffin sections were cut and placed on MAS-coated glass slides (Matsunami Glass Ind. Ltd., Osaka, Japan). Several prepared serial sections were stained with hematoxylin and eosin (HE) to examine age-related morphological changes in the lacrimal gland tissue and toluidine blue (TB) stain following a standard procedure to identify mast cells because mast cells serve as the primary source of histamine in salivary glands [42].
To identify infiltrating cells and ICAM-1 expression in aged mice, immunohistochemical analysis was performed. The sections were deparaffinized and fixed in 0.3% H2O2 and methanol for 30 min to quench endogenous peroxidases. The sections were incubated in citrate buffer (pH 6.0) for 60 min at 65 °C for antigen retrieval. The sections were rinsed with PBS and incubated with 5% normal goat serum in PBS, followed by incubation with purified anti-CD3e, CD4, CD8α, B220, CD11c, F4/80, and ICAM-1 (1:400) at 4 °C overnight. After several rinses with PBS, the sections were incubated with horseradish peroxidase-conjugated goat anti-rat and anti-rabbit antibodies. After washing, the sections were incubated with the detection mixture from the DAB Detection Kit (Nichirei, Tokyo, Japan). Hematoxylin was used as the counterstain.

4.4. RNA Extraction and RT-qPCR

RT-qPCR analyses were performed to investigate age-associated changes in the mRNA expression of HDC, histamine receptors (H1, H2, and H4), inflammatory cytokines (TNF-α, IL-1β and IL-6), anti-inflammatory factor (PPARγ), and ICAM-1 in the lacrimal gland. Total RNA was isolated from the lacrimal glands using NucleoSpin (Takara, Shiga, Japan). Total RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara, Shiga, Japan). qRT-PCR was performed using a StepOneTM Real-Time PCR Thermal Cycler (Applied Biosystems). The following program was used: 50 cycles at 95 °C for 5 s and 60 °C for 30 s. The Ct values obtained from actin expression were used for normalization. The primers used for RT-qPCR are presented in Table 1.

4.5. Histological Quantification and Statistical Analysis

HE-stained TB-stained sections were used for the qualitative assessment of cell infiltration and mast cells, respectively. Cell infiltration counts were taken randomly at middle magnifications (with original magnification of 10×) for five different HE-stained sections from the same specimen; cell infiltration in which more than 50 infiltrating mononuclear cells within five 1 mm × 1 mm frames (1 mm2) were counted, and their average was calculated. For the quantification of mast cells, the number of metachromatic cells observed in TB-stained sections were counted using the same method as the HE-stained sections, and the average was determined. The evaluation was conducted independently by two researchers.
For quantitative data analysis, a t-test was used to determine the differences between paired samples (Microsoft Excel analysis tool). Statistical significance was set at p < 0.05. All data are presented as mean ± standard error of the mean.

5. Conclusions

In summary, we demonstrated that in C57BL/6 wild-type mice, aging is associated with upregulated expression of HDC and histamine H1 receptor, accompanied by lymphocytic infiltration predominantly consisting of T- and B-lymphocytes and increased expression of the pro-inflammatory cytokines TNF-α and IL-1β. Furthermore, these age-related inflammatory changes were attenuated in the HDC-KO mice. A similar trend was observed in the expression of ICAM-1, an adhesion molecule that is strongly associated with inflammatory cell migration.
In the present study, we demonstrated that histamine, as a trigger molecule, may be associated with age-related inflammation through symptom analysis. To the best of our knowledge, this is the first report to demonstrate an association between age-related inflammation of the lacrimal gland and HDC. The findings of the present study may contribute to a better understanding of the underlying mechanisms and development of therapeutic strategies for autoimmune inflammatory diseases such as Sjögren’s syndrome.

Author Contributions

Conceptualization, H.O.; methodology, H.O. and Y.T.; software, H.O.; formal analysis, H.O. and Y.T.; data curation, H.O.; writing—original draft preparation, H.O. and M.K.; writing—review and editing, S.S.; project administration: N.N. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by JSPS KAKENHI Grants (Number 22K09933) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Institutional Review Board Statement

All experiments were performed with the approval of the Animal Care Committee of Nippon Veterinary and Life Science University (Permit Number: 2024K-1 and 2025K-1; Approval Date: 1 April 2024 and 31 March 2025), following all applicable international, national, and institutional guidelines for the care and use of animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We gratefully acknowledge the work of the past and present members of our laboratory for their helpful discussions and comments on this manuscript. We thank Endo, Y. Division of Oral and Maxillofacial Surgery, Graduate School of Dentistry, Tohoku University, and Ohtsu, H. Tekiju Rehabilitation Hospital, for providing HDC-deficient animals. This study was supported by JSPS KAKENHI Grants (Number 22K09933) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
HDCHistidine decarboxylase
RT-qPCRReverse transcriptase-quantitative polymerase chain reaction
ICAM-1Intercellular adhesion molecule-1
TNFαTumor necrosis factor alpha
IL-1βInterleukin-1 beta
IL-6Interleukin-6
LPSLipopolysaccharide
NBPNitrogen-containing bisphosphonate
TBTolidine blue
HEHematoxylin and eosin
PPARγPeroxisome proliferator-activated receptor gamma
PBSPhosphate-buffered saline

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Figure 1. Expression of HDC and histamine receptors in the lacrimal glands of 6-week-old (6 w) and 12-month-old (12 M) wild-type mice. Relative expression was estimated using the expression in 6-week-old mice (A,C–E) or 6-week-old male mice (B) as a control. (A) HDC mRNA expression in the lacrimal glands. (B) HDC expression in the lacrimal glands of male and female mice. (C) Relative mRNA expression of H1 (Hrh1) in the lacrimal glands. (D) Relative mRNA expression of H2 (Hrh2) in lacrimal glands. (E) Relative mRNA expression of H4 (Hrh4) in the lacrimal glands. Asterisks indicate statistical significance (* p < 0.05). Error bars represent the standard error of the mean (SEM). NS means “not statistically significant.” (A,C–E): n = 20 (10 males and 10 females) in each group. (B) n = 10 in each sex.
Figure 1. Expression of HDC and histamine receptors in the lacrimal glands of 6-week-old (6 w) and 12-month-old (12 M) wild-type mice. Relative expression was estimated using the expression in 6-week-old mice (A,C–E) or 6-week-old male mice (B) as a control. (A) HDC mRNA expression in the lacrimal glands. (B) HDC expression in the lacrimal glands of male and female mice. (C) Relative mRNA expression of H1 (Hrh1) in the lacrimal glands. (D) Relative mRNA expression of H2 (Hrh2) in lacrimal glands. (E) Relative mRNA expression of H4 (Hrh4) in the lacrimal glands. Asterisks indicate statistical significance (* p < 0.05). Error bars represent the standard error of the mean (SEM). NS means “not statistically significant.” (A,C–E): n = 20 (10 males and 10 females) in each group. (B) n = 10 in each sex.
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Figure 2. Toluidine Blue (TB) staining and mast cell counts of the lacrimal gland in 6-week- and 12-month-old wild-type mice. (A) A lacrimal gland of 6-week-old wild-type mice. (B) A lacrimal gland of 12-month-old wild-type mice. (C) Number of mast cells per unit area in the lacrimal glands of 6-week-old (6 w) and 12-month-old (12 M) wild-type mice. (D) Number of mast cells per unit area in the lacrimal glands of 12-month-old male and female mice. Scale bars represent 75 µm. Arrows indicate metachromatic reactions. Asterisks indicate statistical significance (* p < 0.05). Error bars represent SEM. NS means “not statistically significant”. (A–C) n = 20 (10 males and 10 females) in each group. (D) n = 10 in each sex.
Figure 2. Toluidine Blue (TB) staining and mast cell counts of the lacrimal gland in 6-week- and 12-month-old wild-type mice. (A) A lacrimal gland of 6-week-old wild-type mice. (B) A lacrimal gland of 12-month-old wild-type mice. (C) Number of mast cells per unit area in the lacrimal glands of 6-week-old (6 w) and 12-month-old (12 M) wild-type mice. (D) Number of mast cells per unit area in the lacrimal glands of 12-month-old male and female mice. Scale bars represent 75 µm. Arrows indicate metachromatic reactions. Asterisks indicate statistical significance (* p < 0.05). Error bars represent SEM. NS means “not statistically significant”. (A–C) n = 20 (10 males and 10 females) in each group. (D) n = 10 in each sex.
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Figure 3. Hematoxylin and Eosin (HE) staining of the lacrimal gland in 6-week and 12-month-old wild-type (WT) and HDC-KO mice. (A) A lacrimal gland of 6-week-old wild-type mice. (B) A lacrimal gland of 12-month-old wild-type mice. (C) A lacrimal gland of 6-week-old HDC-KO mice. (D) A lacrimal gland of 12-month-old HDC-KO mice. (E) Number of cell infiltration per unit area in the lacrimal glands of 12-month-old wild-type and HDC-KO mice. (F) Number of cell infiltration per unit area in the lacrimal glands of 12-month-old male and female wild-type and HDC-KO mice. Scale bars indicate 100 μm. Arrow shows cell infiltration. Open bars and black bars; wild type and HDC-KO, respectively. Asterisks indicate statistical significance (* p < 0.05). Error bars represent SEM. NS means “not statistically significant”. (A–E) n = 20 (10 males and 10 females) in each group. (F) n = 10 in each group.
Figure 3. Hematoxylin and Eosin (HE) staining of the lacrimal gland in 6-week and 12-month-old wild-type (WT) and HDC-KO mice. (A) A lacrimal gland of 6-week-old wild-type mice. (B) A lacrimal gland of 12-month-old wild-type mice. (C) A lacrimal gland of 6-week-old HDC-KO mice. (D) A lacrimal gland of 12-month-old HDC-KO mice. (E) Number of cell infiltration per unit area in the lacrimal glands of 12-month-old wild-type and HDC-KO mice. (F) Number of cell infiltration per unit area in the lacrimal glands of 12-month-old male and female wild-type and HDC-KO mice. Scale bars indicate 100 μm. Arrow shows cell infiltration. Open bars and black bars; wild type and HDC-KO, respectively. Asterisks indicate statistical significance (* p < 0.05). Error bars represent SEM. NS means “not statistically significant”. (A–E) n = 20 (10 males and 10 females) in each group. (F) n = 10 in each group.
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Figure 4. Immunohistochemical staining of the lacrimal gland in 12-month-old wild-type mice. (A) Immunohistochemistry for CD3ε. (B) Immunohistochemistry for CD4. (C) Immunohistochemistry for CD8α. (D) Immunohistochemistry for B220. (E) Immunohistochemistry for CD11c. (F) Immunohistochemistry for F4/80. Scale bars indicate 25 µm. Arrows indicate positive reactions. (A–F) n = 20 (10 males and 10 females).
Figure 4. Immunohistochemical staining of the lacrimal gland in 12-month-old wild-type mice. (A) Immunohistochemistry for CD3ε. (B) Immunohistochemistry for CD4. (C) Immunohistochemistry for CD8α. (D) Immunohistochemistry for B220. (E) Immunohistochemistry for CD11c. (F) Immunohistochemistry for F4/80. Scale bars indicate 25 µm. Arrows indicate positive reactions. (A–F) n = 20 (10 males and 10 females).
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Figure 5. Pro-inflammatory cytokines and PPAR mRNA expression in the lacrimal glands in 6-week and 12-month-old wild-type (WT) and HDC-KO mice. Relative expression was estimated using the expression in 6-week-old wild-type control mice as the control Ct value. (A) TNF-α mRNA expression in the lacrimal glands. (B) IL-1β mRNA expression in the lacrimal glands. (C) Il-6 mRNA expression in the lacrimal glands. (D) PPARγ mRNA expression in the lacrimal glands. Open bars and black bars; wild type and HDC-KO, respectively. Asterisks indicate statistical significance (* p < 0.05). Error bars represent SEM. NS and ND mean “not statistically significant” and “Not detected”, respectively. (A–D) n = 20 (10 males and 10 females) in each group.
Figure 5. Pro-inflammatory cytokines and PPAR mRNA expression in the lacrimal glands in 6-week and 12-month-old wild-type (WT) and HDC-KO mice. Relative expression was estimated using the expression in 6-week-old wild-type control mice as the control Ct value. (A) TNF-α mRNA expression in the lacrimal glands. (B) IL-1β mRNA expression in the lacrimal glands. (C) Il-6 mRNA expression in the lacrimal glands. (D) PPARγ mRNA expression in the lacrimal glands. Open bars and black bars; wild type and HDC-KO, respectively. Asterisks indicate statistical significance (* p < 0.05). Error bars represent SEM. NS and ND mean “not statistically significant” and “Not detected”, respectively. (A–D) n = 20 (10 males and 10 females) in each group.
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Figure 6. Immunohistochemistry and qPCR for ICAM-1 in lacrimal glands. (A) Immunohistochemistry for ICAM-1 in a lacrimal gland of 12-month-old wild-type mice. (B) Immunohistochemistry for ICAM-1 in a lacrimal glands of 12-month-old HDC-KO mice. (C) Quantitative RT-PCR to determine ICAM-1 mRNA expression in the lacrimal glands of 6-week (6 w) and 12-month-old (12 M) wild-type and HDC-KO mice. Scale bars represent 50 μm. Arrows indicate positive reactions. Open and black bars represent wild-type (WT) and HDC-KO mice, respectively. Asterisks indicate statistical significance. Error bars represent SEM. NS means “not statistically significant.” (A–C) n = 20 (10 males and 10 females) in each group.
Figure 6. Immunohistochemistry and qPCR for ICAM-1 in lacrimal glands. (A) Immunohistochemistry for ICAM-1 in a lacrimal gland of 12-month-old wild-type mice. (B) Immunohistochemistry for ICAM-1 in a lacrimal glands of 12-month-old HDC-KO mice. (C) Quantitative RT-PCR to determine ICAM-1 mRNA expression in the lacrimal glands of 6-week (6 w) and 12-month-old (12 M) wild-type and HDC-KO mice. Scale bars represent 50 μm. Arrows indicate positive reactions. Open and black bars represent wild-type (WT) and HDC-KO mice, respectively. Asterisks indicate statistical significance. Error bars represent SEM. NS means “not statistically significant.” (A–C) n = 20 (10 males and 10 females) in each group.
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Table 1. Sequence of primers used in this study for RT-qPCR.
Table 1. Sequence of primers used in this study for RT-qPCR.
NameSequence
HdcF: 5′-TTCCAGCCTCCTCTGTCTGT-3′
R: 5′-GGTATCCAGGCTGCACATTT-3′
Hrh1F: 5′-GGGAAAHHHAAACAGTCACA-3′
R: 5′-ACTGTCGATCCACCAAGGTC-3′
Hrh2F: 5′-CAGCTTCCATCCTCAACCTC-3′
R: 5′-GACCTGCACTTTGCACTTGA-3′
Hrh4F: 5′-GAATCAGCTGCATCTCGTCA-3′
R: 5′-GTGACCTGGCTAGCTTCCTG-3′
TnfaF: 5′-TATGGCTCAGGGTCCAACTC-3′
R: 5′-CTCCCTTTGCAGAACTCAGG-3′
Il1bF: 5′-GCCCATCCTCTGTGACTCAT-3′
R: 5′-AGGCCACAGGTATTTTGTCG-3′
Il6F: 5′-AGTTGCCTTCTTGGGACTGA-3′
R: 5′-TCCACGATTTCCCAGAGAAC-3′
PparγF: 5′-TTTTCAAGGGTGCCAGTTTC-3′
R: 5′-AATCCTTGGCCCTCTGAGAT-3′
Icam-1F: 5′-AGCACCTCCCCACCTACTTT-3′
R: 5′-AGCTTGCACGACCCTTCTAA-3′
ActbF: 5′-GCGTGACATTAAAGAGAAGCTG-3′
R: 5′-CTCAGGAGGAGCAATGATCTTG-3′
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MDPI and ACS Style

Otsuka, H.; Tsunoyama, Y.; Koh, M.; Soeta, S.; Nonaka, N. Histamine Deficiency Inhibits Lymphocyte Infiltration in the Lacrimal Gland of Aged Mice. Lymphatics 2025, 3, 48. https://doi.org/10.3390/lymphatics3040048

AMA Style

Otsuka H, Tsunoyama Y, Koh M, Soeta S, Nonaka N. Histamine Deficiency Inhibits Lymphocyte Infiltration in the Lacrimal Gland of Aged Mice. Lymphatics. 2025; 3(4):48. https://doi.org/10.3390/lymphatics3040048

Chicago/Turabian Style

Otsuka, Hirotada, Yusuke Tsunoyama, Miki Koh, Satoshi Soeta, and Naoko Nonaka. 2025. "Histamine Deficiency Inhibits Lymphocyte Infiltration in the Lacrimal Gland of Aged Mice" Lymphatics 3, no. 4: 48. https://doi.org/10.3390/lymphatics3040048

APA Style

Otsuka, H., Tsunoyama, Y., Koh, M., Soeta, S., & Nonaka, N. (2025). Histamine Deficiency Inhibits Lymphocyte Infiltration in the Lacrimal Gland of Aged Mice. Lymphatics, 3(4), 48. https://doi.org/10.3390/lymphatics3040048

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