J Korean Med Sci.  2006 Jun;21(3):391-396. 10.3346/jkms.2006.21.3.391.

B Cells in Murine Cervical Lymph Nodes are Conventional B-2 Cells

• Yeo SG
• Cho JS
• Park DC

• KMID: 1778417
• DOI: http://doi.org/10.3346/jkms.2006.21.3.391

Abstract

We investigated the characteristic features of cervical lymph node B cells to determine whether their behavior differs from that of B cells located elsewhere, because cervical lymph nodes may be exposed to continual antigenic stimulation from the naso- and/or oropharynx. B cells were isolated from cervical lymph nodes, spleen and peritoneal fluid of mice, cultured in medium, and exposed to various stimuli. The expression of various surface molecules characteristic of lymphoid B cells was assayed by flow cytometry, and immunoglobulin secreted into the culture supernatants was evaluated by enzyme-linked immunosorbent assay. B220+ cells were cultured in medium alone or with lipopolysaccharide, and their entrance into S phase in response to stimuli was measured by proliferative assays. Phenotypic characteristics of cervical lymph node B cells included CD5low, CD23high, CD43low, B7.1low, B7.2low, and Syndecan-1low. Unstimulated lymphoid B cells did not secrete immunoglobulin, but, upon stimulation, secretion of IgM was increased more than secretion of IgA and IgG. B cells actively entered S phase after 48 hr stimulation. These results show that B cells in cervical lymph nodes are conventional B2 cells, like splenic B cells.

Keyword

B-Lymphocytes; Lymph Nodes; Flow Cytometry; Enzyme-linked Immunosorbent Assay; Cell Proliferation

MeSH Terms

Spleen/cytology
Phenotype
Mice, Inbred BALB C
Mice
Male
Lymph Nodes/*cytology
Immunoglobulin M/chemistry
Flow Cytometry
Enzyme-Linked Immunosorbent Assay
Culture Media/pharmacology
Cell Proliferation
Cell Culture Techniques/methods
B-Lymphocytes/*cytology
Antigens/metabolism
Animals

Figure

• 

  Fig. 1 Antigen surface expression on (A) cervical lymph node (B) spleen (C) peritoneal fluid B cells. B cells of BALB/cByJ mice were harvested and stained with Abs specific for B220, CD5, CD23, CD43, B7.1, B7.2, IgM, and Syndacan-1. Expression of functional molecules on B cells was analyzed by two-color flow cytometry.

• 

  Fig. 2 Survival rates of stimulated and unstimulated B cells.

• 

  Fig. 3 IgM production by murine cervical lymph node B cells. Cells (0.5×106per group) were cultured in fetal bovine serum (FBS)-containing culture medium with or without stimulation for 5 days. Supernatants were collected and assayed for IgM secretion using an enzyme-linked immunosorbent assay (ELISA).

• 

  Fig. 4 IgM production by peritoneal B cells. Assays were performed as shown in the legend to Fig. 5.

• 

  Fig. 5 Stimulation of B cell entry into S phase. Primary murine B cells were cultured for 24 hr and 48 hr with medium alone, LPS (25 µg/mL), CD40L (1:10) plus anti-CD8 antibody (1:40), or interleukin (IL)-4 (50 U/mL). [3H] thymidine was added during the last 6 hr in culture, and incorporation of label was assessed. Mean values of quadruplicate cultures are shown along with lines indicating the standard errors of the means.

Reference

1. Kim JB, Yeo SG, Kim SW, Cho JS, Cha CI. Characteristic features of immune B cells in murine cervical lymph node. Korean J Otolaryngol. 2005. 48:241–246.

2. Rothstein TL. Cutting Edge: Commentary: Two B-1 or Not To Be One. J Immunol. 2002. 168:4257–4261.

3. Hayakawa K, Hardy RR. Development and function of B-1 cells. Curr Opin Immunol. 2000. 12:346–353.

4. Beckstead JH. The evaluation of human lymph nodes, using plastic sections and enzyme histochemistry. Am J Clin Pathol. 1983. 80:131–139.

5. Hsu SM, Cossman J, Jaffe ES. Lymphocyte subsets in normal human lymphoid tissues. Am J Clin Pathol. 1983. 80:21–30.

6. Gavin MF, Laura AS, William DH, Kejian Y, Rachel MG, Babara SN. Splenic and peritoneal B-1 cells differ in terms of transcriptional and proliferative features that separate peritoneal B-1 from splenic B-2 cells. Cellular Immunol. 2001. 213:62–71.

7. Yeo SG, Cho JS, Park DC, Rothstein TL. B-1 cells differ from conventional B (B-2) cells. Immune Network. 2004. 4:155–160.

8. Martin F, Kearney JF. B-1 cells: similarities and differences with other B cell subsets. Curr Opin Immunol. 2001. 195–201.

9. Robert B, Henry HW. Origins and functions of B-1 cells with notes on the role of CD5. Annu Rev Immunol. 2002. 20:253–300.

10. Casola S, Otipoby KL, Alimzhanov M. B cell receptor signal strength determines B cell fate. Nat Immunol. 2004. 5:317–327.

11. Barr TA, Carlring J, Heath AW. CD40 antibody provides the CD40 signal to B cells, but does not substitute for T cell help in responses to TD antigens. Vaccine. 2005. 23:3477–3482.

12. Davey EJ, Thyberg J, Conrad D, Severinson E. Expression and production of interleukin 4 in B-cell chronic lymphocytic leukaemia. Leuk Lymphoma. 2001. 42:689–698.

13. Kerr WG, Heller M, Herzenberg LA. Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains. Proc Natl Acad Sci USA. 1996. 93:3947–3952.

14. Beisner DR, Chen IL, Kolla RV, Hoffmann A. Cutting edge: innate immunity conferred by B cells is regulated by caspase-8. J Immunol. 2005. 175:3469–3473.

15. Segura AS, Brieva JA, Rodriguez C. Regualtion of immunoglobulin secretion of plasma cells infiltrating nasal polyps. Laryngoscope. 2000. 110:1183–1188.

16. Suenaga S, Kodama S, Ueyama S, Suzuki M, Mogi G. Mucosal immunity of the middle ear: Analysis at the single cell level. Laryngoscope. 2001. 111:290–296.

17. Arita M, Kodama S, Suzuki M, Mogi G. Single cell analysis of adenoid CD5+B cells and their protective contributions to nasopharyngeal immunity. Laryngoscope. 2003. 113:484–491.

18. Marianne B, Andrey PP, Tara S, Michael CC, Jianzhu C. A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection. J Exp Med. 1998. 12:2381–2386.

19. Piatelli MJ, Tanguay D, Rothstein TL, Chiles TC. Cell cycle control mechanism in B-1 and B-2 lymphoid subsets. Immunol Res. 2003. 27:31–52.

20. Tanguay DA, Colarusso TP, Doughty C, Pavlovic-Ewer S, Rothstein TL, Chiles TC. Cutting edge: Differential signaling requirements for activation of assembled cyclinD3-cdk4 complexes in B-1 and B-2 lymphocyte subsets. J Immunol. 2001. 166:4273–4277.