Characterization of rag1 mutant zebrafish leukocytes

Abstract

Background: Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants).

Results: Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte-like cells and comprised 7% of the gated cells in the rag1 mutants and 26% in the wild-types. Reverse transcriptase polymerase chain reaction (RT-PCR) assays demonstrated rag1 mutant kidney hematopoietic tissue expressed mRNA encoding Non-specific Cytotoxic cell receptor protein-1 (NCCRP-1) and Natural Killer (NK) cell lysin but lacked T cell receptor (TCR) and immunoglobulin (Ig) transcript expression, while wild-type kidney hematopoietic tissue expressed NCCRP-1, NK lysin, TCR and Ig transcript expression.

Conclusion: Our study demonstrates that in comparison to wild-type zebrafish, rag1 mutants have a significantly reduced lymphocyte-like cell population that likely includes Non-specific cytotoxic cells (NCC) and NK cells (and lacks functional T and B lymphocytes), a similar macrophage/monocyte population, and a significantly increased neutrophil population. These zebrafish have comparable leukocyte populations to SCID and rag 1 and/or 2 mutant mice, that possess macrophages, natural killer cells and neutrophils, but lack T and B lymphocytes. Rag1 mutant zebrafish will provide the platform for remarkable investigations in fish and innate immunology, as rag 1 and 2 mutant mice did for mammalian immunology.

Figure 1

Mutant and wild-type zebrafish leukocyte differentials and flow cytometry scatter plots. I and II represent differential counts on peripheral blood smears and kidney hematopoietic tissue smears, respectively. Asterisks indicate significant difference between wild-type and mutant blood cells within the specific population. Average percentage ± standard deviation from 10 replicates is shown (p ≤ 0.05). III and IV show graphs of flow cytometric results on cells from mutant and wild-type kidney tissues, respectively. Graphs represent pooled data from 4 runs on 4 separate mutant and wild-type fish. Forward scatter (FSC) and side scatter (SSC) analyses of whole kidney cell suspension differentiates three distinct cell populations: A-macrophage/monocytes and granulocytes, B-hematopoietic precursor cells and C-lymphocytes and lymphocyte-like cells. Note the reduction in gate C, characteristic of lymphocytes and lymphocyte-like cells, in mutant kidney cell suspensions.

Figure 2

RT-PCR analyses of mutant and wild-type zebrafish kidney hematopoietic tissue. Evaluation of expression of Immunoglobulin (Ig) heavy chain gene rearrangements, T-cell receptor (TCR) β chain rearrangements, NK-Lysin, NCC Receptor Protein-1 (NCCRP-1), and transcription elongation factor 1-α (EF1-α, as a positive control) in mutant and wild-type zebrafish by RT-PCR. (A) Nested RT-PCR using primers spanning VDJ-Cm in Ig VH1-VH4 [16]. (B) Nested RT-PCR using primers spanning TCR V(D)J-C_β [16]. (C) RT-PCR was used to analyze mRNA expression using primers specific for NCCRP-1, NK lysin form 1, NK lysin forms 2 and 3 and EF1-α (primers are listed in Table 1). All RT-PCR assays included no-RT controls and no product was obtained (data not shown).